Nucleases and methods for making and using them

ABSTRACT

Disclosed herein are polypeptides having nuclease activity. Some of the polypeptides having nuclease activity were generated by an improved gene site mutagenesis (“GSSM”) method or the tailored multi-site combinatorial assembly (“TMCA”) method. Also disclosed are compositions and kits comprising the polypeptides having nuclease activity, and methods for making and using these polypeptides, compositions and kits.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBASF_060_PR_SEQLISTING.TXT, created Dec. 20, 2017, which is 4 Kb insize. The information in the electronic format of the Sequence Listingis incorporated herein by reference in its entirety.

BACKGROUND Field

The present disclosure relates generally to molecular and cellularbiology and biochemistry. More specifically, the disclosure relates topolypeptides having nuclease activity, polynucleotides comprising thecoding sequences for these polypeptides, and methods for making andusing these polypeptides and polynucleotides.

Description of the Related Art

Nucleases are enzymes capable of cleaving the phosphodiester bondsbetween monomers of nucleic acids, so that they can process long-chainof nucleic acids into smaller units. Nucleases have been used widely inindustrial and pharmaceutical applications. For example, the use ofnucleases in fermentation processes can remove DNA, and facilitate andimprove the production and recovery of proteins and other molecules ofinterest. DNA removal is important in fermentation processes since manyof those processed utilize recombinant DNA technologies, and manyapplicable regulations require the confirmation of an absence of rDNAwhich can be removed by nucleases. Nucleases can also be used forbiofilm removal, which has multiple applications, for example cleaningfood machinery, hard surface cleaning, paper machines, wound healing,and oral care. There is a need for generating effective nucleases havingdesired optimal temperature and pH range.

SUMMARY

Disclosed herein includes polypeptides having nuclease activity(hereinafter “nucleases” or “nuclease polypeptides”), polynucleotidescomprising the coding sequences for these polypeptides (hereafter“nuclease polynucleotides), and methods for making and using thesepolypeptides and polynucleotides. Also provided herein are compositionsand kits comprising one or more of the nuclease polypeptides disclosedherein, one or more of the nuclease-coding polynucleotides, and anycombination thereof.

Disclosed herein includes synthetic or recombinant polypeptidecomprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 1. In some embodiments, the polypeptide has nucleaseactivity, and wherein the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, V226K, E230R,G263A, G119N, V226K, G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K,P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N,P84L, S74N, T82R, G75R, Q141R, and D107N. In some embodiments, the aminoacid sequence has at least 90% sequence identity to SEQ ID NO: 1. Insome embodiments, the polypeptide comprises one or more mutationsselected from the group consisting of L114F, G119N, D107N, S74N, Q141R,T82R, and G75R. In some embodiments, the polypeptide comprises acombination of mutations selected from the group consisting of:

-   -   (T82R, L114F);    -   (T82R, L114F, G119N);    -   (T82R)    -   (T82R, G119N);    -   (L114F);    -   (L114F, G119N);    -   (G119N);    -   (G75R, T82R, L114F, G119N);    -   (G75R, T82R, L114F, G119N);    -   (G75R, T82R);    -   (G75R, L114F);    -   (G75R, L114F, G119N);    -   (S74N, G75R, T82R, G119N, Q141R);    -   (S74N, T82R, L114F, G119N);    -   (S74N, T82R, L114F, G119N, Q141R);    -   (S74N, T82R, Q141R);    -   (S74N, T82R, G119N, Q141R);    -   (S74N, L114F, Q141R);    -   (S74N, L114F, G119N, Q141R);    -   (S74N, Q141R);    -   (S74N, G75R, T82R, L114F, Q141R);    -   (S74N, G75R, T82R, L114F, G119N, Q141R);    -   (S74N, G75R, T82R, Q141R);    -   (S74N, G75R, L114F, Q141R);    -   (S74N, G75R, Q141R);    -   (T82R, D107N);    -   (G75R, T82R, D107N);    -   (D107N);    -   (G75R, T82R, G119N);    -   (T82R, D107N, G119N);    -   (G75R, T82R, D107N, G119N);    -   (D107N, G119N);    -   (G75R, D107N, G119N);    -   (S74N, T82R, D107N, Q141R);    -   (S74N, G75R, T82R, D107N, Q141R);    -   (S74N, G75R, D107N, Q141R);    -   (S74N, G119N, Q141R);    -   (S74N, G75R, G119N, Q141R);    -   (S74N, T82R, D107N, G119N, Q141R);    -   (S74N, G75R, T82R, D107N, G119N, Q141R);    -   (S74N, D107N, G119N, Q141R);    -   (S74N, G75R, D107N, G119N, Q141R);    -   (T82R, D107N, L114F);    -   (G75R, T82R, D107N, L114F);    -   (D107N, L114F);    -   (G75R, D107N, L114F);    -   (G75R, D107N);    -   (T82R, D107N, L114F, G119N);    -   (D107N, L114F, G119N);    -   (G75R, D107N, L114F, G119N);    -   (S74N, T82R, D107N, L114F, Q141R);    -   (S74N, G75R, T82R, D107N, L114F, Q141R);    -   (S74N, D107N, L114F, Q141R);    -   (S74N, G75R, D107N, L114F, Q141R);    -   (S74N, D107N, Q141R);    -   (S74N, G75R, L114F, G119N, Q141R);    -   (S74N, T82R, D107N, L114F, G119N, Q141R);    -   (S74N, G75R, T82R, D107N, L114F, G119N, Q141R);    -   (S74N, D107N, L114F, G119N, Q141R);    -   (S74N, G75R, D107N, L114F, G119N, Q141R); and    -   (G75R, T82R, D107N, L114F, G119N).

In some embodiments, the amino acid sequence of the polypeptide differsfrom the amino acid sequence of SEQ ID NO: 1 for, or only for,comprising one or more mutations of E230L, L114F, E260R, E230M, D246P,S161R, N54S, T227H, V226K, E230R, G263A, G119N, V226K, G263A, N127S,P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S, A73M, P179L,Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R, G75R, Q141R,and D107N.

In some embodiments, the polypeptide is more thermotolerant compared tothe nuclease having the sequence of SEQ ID NO: 1. In some embodiments,the nuclease activity of the polypeptide is at least 5% higher than thatof the nuclease having the sequence of SEQ ID NO: 1 at 10° C. to 70° C.In some embodiments, the nuclease activity of the polypeptide is atleast 5% higher than that of the nuclease having the sequence of SEQ IDNO: 1 at 37° C. to 60° C. In some embodiments, the nuclease activity ofthe polypeptide is at least 5% higher than that of the nuclease havingthe sequence of SEQ ID NO: 1 at 40° C. to 60° C. In some embodiments,the nuclease activity ratio 54° C./37° C. of the polypeptide is at least5% higher than that of the nuclease having the sequence of SEQ ID NO: 1.In some embodiments, the optimal temperature of the polypeptide isbetween 45° C. to 55° C. In some embodiments, the optimal pH of thepolypeptide is between pH 4 to pH 11. In some embodiments, thepolypeptide comprises no signal sequence. In some embodiments, thepolypeptide comprises a signal sequence. In some embodiments, the signalsequence is a heterologous sequence.

Also disclosed herein includes a composition comprising one or more ofthe polypeptides disclosed herein. In some embodiments, the compositionis a reaction mixture, a detergent composition, a detergent additive, afood, a food supplement, a feed supplement, a feed, a pharmaceuticalcomposition, a fermentation product, a fermentation intermediate, afermentation downstream reaction mixture, a reaction mixture, or acombination thereof. In some embodiments, the reaction mixturecomprising one or more of the polypeptides is for protein expression orpurification. For example, the polypeptide can be a polypeptidecomprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 1, where the polypeptide has nuclease activity, and wherethe polypeptide comprises one or more mutations of E230L, L114F, E260R,E230M, D246P, S161R, N54S, T227H, V226K, E230R, G263A, G119N, V226K,G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S,A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R,G75R, Q141R, and D107N.

Also disclosed herein includes a synthetic or recombinant nucleic acidthat encodes any one of the polypeptides disclosed herein, and anexpression vector comprising the polynucleotide sequence of thesynthetic or recombinant nucleic acid. In some embodiments, theexpression vector comprises a viral vector, a plasmid, a phage, aphagemid, a cosmid, a fosmid, a bacteriophage, an artificial chromosome,or a combination thereof. For example, the polypeptide can be apolypeptide comprising an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 1, where the polypeptide has nucleaseactivity, and where the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, V226K, E230R,G263A, G119N, V226K, G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K,P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N,P84L, S74N, T82R, G75R, Q141R, and D107N.

Disclosed herein includes a recombinant cell comprising one or more ofthe polypeptides disclosed herein, one or more synthetic or recombinantnucleic acid that encodes any one of the polypeptides disclosed herein,one or more expression vectors comprising the polynucleotide sequence ofthe synthetic or recombinant nucleic acid, or a combination thereof. Insome embodiments, the nucleic acid is a part of a chromosome of therecombinant cell. In some embodiments, the recombinant cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insectcell, or a plant cell. For example, the polypeptide can be a polypeptidecomprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 1, where the polypeptide has nuclease activity, and wherethe polypeptide comprises one or more mutations of E230L, L114F, E260R,E230M, D246P, S161R, N54S, T227H, V226K, E230R, G263A, G119N, V226K,G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S,A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R,G75R, Q141R, and D107N.

Disclosed herein includes a method of producing a recombinantpolypeptide having nuclease activity. The method, in some embodiments,comprises: expressing the nucleic acid that encodes any one of thepolypeptides disclosed herein under conditions that allow expression ofthe polypeptide, thereby producing recombinant polypeptide havingnuclease activity, wherein the nucleic acid is operably linked to apromoter. In some embodiments, the nucleic acid is present in anexpression vector. In some embodiments, the nucleic acid is present in ahost cell to allow expression of the polypeptide. In some embodiments,the nucleic acid is present in a chromosome of the host cell. In someembodiments, the host cell is a cell from an organism selected from thegroup consisting of Pichia pastoris (Komagataella pastoris), Bacillussubtilis, Pseudomonas fluorescens, Myceliopthora thermophile fungus,Tricodermea reesei, Escherichia coli, Bacillus licheniformis,Aspergillus niger, Schizosaccharomyces pombe, and. Sacaramycescerevisiae. In some embodiments, the nucleic acid is present an in vitroexpression system. The polypeptide can be, in some embodiments, apolypeptide comprising an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 1, where the polypeptide has nucleaseactivity, and where the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, V226K, E230R,G263A, G119N, V226K, G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K,P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N,P84L, S74N, T82R, G75R, Q141R, and D107N.

Disclosed herein includes a method for degrading a polynucleotide,comprising contacting a polynucleotide molecule with one or more of thepolypeptides disclosed herein, thereby degrading the polynucleotidemolecule. For example, the polypeptide can be a polypeptide comprisingan amino acid sequence having at least 80% sequence identity to SEQ IDNO: 1, where the polypeptide has nuclease activity, and where thepolypeptide comprises one or more mutations of E230L, L114F, E260R,E230M, D246P, S161R, N54S, T227H, V226K, E230R, G263A, G119N, V226K,G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S,A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R,G75R, Q141R, and D107N. In some embodiments, the polynucleotide moleculeis a DNA molecule or a RNA molecule. In some embodiments, the contactingoccurs at pH 4 to pH 11. In some embodiments, the reaction mixture has atemperature at about 10° C. to about 70° C. In some embodiments, thecontacting occurs at 40° C. to 60° C.

Disclosed herein includes a method for washing an object. The method, insome embodiments, comprises contacting a composition comprising one ormore of the polypeptides disclosed herein with the object under theconditions sufficient for said washing. In some embodiments, thepolypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 1, wherein the polypeptide has nucleaseactivity, and wherein the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, V226K, E230R,G263A, G119N, V226K, G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K,P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N,P84L, S74N, T82R, G75R, Q141R, and D107N. In some embodiments, theobject is a textile.

Also disclosed herein includes a method for degrading DNA or RNA duringprotein production. The method, in some embodiments, comprises culturinga host cell, wherein the host cell comprises a nucleic acid encoding aprotein of interest; and expressing one or more of the polypeptidesdisclosed herein under conditions that allow degradation of DNA or RNAby the one or more polypeptides. In some embodiments, the host cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, a plantcell, or an insect cell. In some embodiments, the polypeptide isexpressed from an expression vector present in the host cell or thepolypeptide is encoded by a nucleic acid sequence in a chromosome of thehost cell. In some embodiments, the polypeptide is expressed by cellsthat do not express the protein of interest. In some embodiments,expression of one or more of the protein of interest and the polypeptideis inducible or non-inducible. In some embodiments, one or more of thepolypeptides disclosed herein are added externally. For example, thepolypeptide can be a polypeptide comprising an amino acid sequencehaving at least 80% sequence identity to SEQ ID NO: 1, where thepolypeptide has nuclease activity, and where the polypeptide comprisesone or more mutations of E230L, L114F, E260R, E230M, D246P, S161R, N54S,T227H, V226K, E230R, G263A, G119N, V226K, G263A, N127S, P84V, D83E,D28G, V45T, M262V, A190K, P84N, I44R, G256S, A73M, P179L, Q135E, A60P,V247I, G263K, S161E, P72N, P84L, S74N, T82R, G75R, Q141R, and D107N.

Also disclose herein include a reaction mixture, wherein the reactionmixture comprises: (a) one or more of the polypeptides disclosed herein,(b) one or more nucleic acid molecules, and (c) an aqueous solutionwherein the polypeptide hydrolyzes the one or more nucleic acidmolecules. In some embodiments, the one or more nucleic acid moleculescomprise single-stranded DNA molecules, double-stranded DNA molecules,single-stranded RNA molecules, double-stranded RNA molecules, or anycombination thereof. In some embodiments, the one or more nucleic acidmolecules are from a host cell for protein production. In someembodiments, the polypeptide is expressed in a host cell selected fromthe group consisting of a bacterial cell, a mammalian cell, a fungalcell, a yeast cell, and an insect cell. In some embodiments, thereaction mixture has a temperature at about 10° C. to about 70° C. Insome embodiments, the reaction mixture has a temperature at about 45° C.to about 55° C. In some embodiments, the reaction mixture is at about pH4 to about pH 11. In some embodiments, the reaction mixture is at aboutpH 6 to about pH 7. In some embodiments, the aqueous solution is adetergent composition, a detergent additive, a food, a food supplement,a feed supplement, a feed, a pharmaceutical composition, a fermentationproduct, a fermentation intermediate, a fermentation downstream reactionmixture, a product from protein production process, an intermediate fromprotein production process, a protein purification solution, or acombination thereof. For example, the polypeptide can be a polypeptidecomprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 1, where the polypeptide has nuclease activity, and wherethe polypeptide comprises one or more mutations of E230L, L114F, E260R,E230M, D246P, S161R, N54S, T227H, V226K, E230R, G263A, G119N, V226K,G263A, N127S, P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S,A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R,G75R, Q141R, and D107N.

Disclosed herein include a method for degrading DNA or RNA in a proteinproduction mixture. The method comprises, in some embodiments, culturinga host cell which comprises a nucleic acid encoding a protein ofinterest; and expressing one or more of the polypeptides disclosedherein under conditions that allow degradation of DNA or RNA by thepolypeptide. The expression of the polypeptide can be delayed, in someembodiments, until after the production of the protein of interest. Insome embodiments, the expression of the polypeptide is not delayed. Theexpression of the polypeptide can start before, after, or at the sametime with the start of the expression of the protein of interest. Thehost cell can be, for example, a bacterial cell, a mammalian cell, afungal cell, a yeast cell, a plant cell, or an insect cell. In someembodiments, the polypeptide is expressed from an expression vectorpresent in the host cell or the polypeptide is encoded by a nucleic acidsequence in a chromosome of the host cell. In some embodiments, thepolypeptide is expressed by cells that do not express the protein ofinterest. The expression of one or more of the protein of interest andthe polypeptides can be inducible or non-inducible.

Also disclosed herein include a method for degrading DNA or RNA duringprotein production. The method comprises, in some embodiments, culturinga host cell that comprises a nucleic acid encoding a protein ofinterest; and adding a polypeptide disclosed herein under conditionsthat allow degradation of DNA or RNA by the polypeptide. The host cellcan be, for example, a bacterial cell, a mammalian cell, a fungal cell,a yeast cell, a plant cell, or an insect cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protein sequence of the nuclease having the sequence ofSEQ ID NO: 1 and the corresponding nucleic acid coding sequence (SEQ IDNO: 2).

FIG. 2 shows temperature profiling of ten nuclease variants.

FIG. 3 shows pH profiling of ten nuclease variants at 37° C.

FIG. 4 is a plot showing heat kill temp ramp of an amylase.

FIG. 5 shows normalized results of nuclease pretreatment of an amylaseBroth.

DETAILED DESCRIPTION

All patents, applications, published applications and other publicationsreferred to herein are incorporated by reference for the referencedmaterial and in their entireties. If a term or phrase is used herein ina way that is contrary to or otherwise inconsistent with a definitionset forth in the patents, applications, published applications and otherpublications that are herein incorporated by reference, the use hereinprevails over the definition that is incorporated herein by reference.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications, and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferences unless indicated otherwise, expressly or by context. Forexample, “a” dimer includes one or more dimers, unless indicatedotherwise, expressly or by context.

The term “amplification” (“a polymerase extension reaction”) means thatthe number of copies of a polynucleotide is increased.

As used herein, “sequence identity” or “identity” in the context of twoprotein sequences (or nucleotide sequences) includes reference to theresidues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window.

Sequence identity usually is provided as “% sequence identity” or “%identity”. To determine the percent-identity between two amino acidsequences in a first step a pairwise sequence alignment is generatedbetween those two sequences, wherein the two sequences are aligned overtheir complete length (i.e., a pairwise global alignment). The alignmentis generated with a program implementing the Needleman and Wunschalgorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using theprogram “NEEDLE” (The European Molecular Biology Open Software Suite(EMBOSS)) with the programs default parameters (gapopen=10.0,gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for thepurpose of this description is that alignment, from which the highestsequence identity can be determined.

After aligning two sequences, in a second step, an identity value isdetermined from the alignment produced. For purposes of thisdescription, percent identity is calculated by:

%-identity=(identical residues/length of the alignment region which isshowing the respective sequence of this description over its completelength)*100.

Thus, sequence identity in relation to comparison of two amino acidsequences according to this embodiment is calculated by dividing thenumber of identical residues by the length of the alignment region whichis showing the respective sequence of this description over its completelength. This value is multiplied with 100 to give “%-identity”.

For calculating the percent identity of two DNA sequences the sameapplies as for the calculation of percent identity of two amino acidsequences with some specifications.

For DNA sequences encoding for a protein the pairwise alignment shall bemade over the complete length of the coding region from start to stopcodon excluding introns. Introns, present in the other sequence, so thesequence to which the sequence of this description is compared, may alsobe removed for the pairwise alignment. Percent identity is thencalculated by: %-identity=(identical residues/length of the alignmentregion which is showing the coding region of the sequence of thisdescription from start to stop codon excluding introns over its completelength)*100.

For non-protein-coding DNA sequences the pairwise alignment shall bemade over the complete length of the sequence of this description, sopercent identity is calculated by: %-identity=(identical residues/lengthof the alignment region which is showing the sequence of thisdescription over its complete length)*100.

Moreover, the preferred alignment program implementing the Needleman andWunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is “NEEDLE” (TheEuropean Molecular Biology Open Software Suite (EMBOSS)) with theprograms default parameters (gapopen=10.0, gapextend=0.5 andmatrix=EDNAFULL).

Sequences, having identical or similar regions with a sequence of thisdescription, and which shall be compared with a sequence of thisdescription to determine % identity, can easily be identified by variousways that are within the skill in the art, for instance, using publiclyavailable computer methods and programs such as BLAST, BLAST-2,available for example at NCBI.

Variants of the parent enzyme molecules may have an amino acid sequencewhich is at least n percent identical to the amino acid sequence of therespective parent enzyme having enzymatic activity with n being aninteger between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 compared to the full lengthpolypeptide sequence. Preferably, variant enzymes which are n percentidentical when compared to a parent enzyme, have enzymatic activity.

Enzyme variants may be defined by their sequence similarity whencompared to a parent enzyme. Sequence similarity usually is provided as“% sequence similarity” or “%-similarity”. For calculating sequencesimilarity in a first step a sequence alignment has to be generated asdescribed above. In a second step, the percent-similarity has to becalculated, whereas percent sequence similarity takes into account thatdefined sets of amino acids share similar properties, e.g., by theirsize, by their hydrophobicity, by their charge, or by othercharacteristics. Herein, the exchange of one amino acid with a similaramino acid is called “conservative mutation”. Enzyme variants comprisingconservative mutations appear to have a minimal effect on proteinfolding resulting in certain enzyme properties being substantiallymaintained when compared to the enzyme properties of the parent enzyme.

For determination of %-similarity according to this description thefollowing applies, which is also in accordance with the BLOSUM62 matrixas for example used by program “NEEDLE”, which is one of the most usedamino acids similarity matrix for database searching and sequencealignments.

Amino acid A is similar to amino acids S;Amino acid D is similar to amino acids E; N;Amino acid E is similar to amino acids D; K; Q;Amino acid F is similar to amino acids W; Y;Amino acid H is similar to amino acids N; Y;Amino acid I is similar to amino acids L; M; V;Amino acid K is similar to amino acids E; Q; R;Amino acid L is similar to amino acids I; M; V;Amino acid M is similar to amino acids I; L; V;Amino acid N is similar to amino acids D; H; S;Amino acid Q is similar to amino acids E; K; R;Amino acid R is similar to amino acids K; Q;Amino acid S is similar to amino acids A; N; T;Amino acid T is similar to amino acids S;Amino acid V is similar to amino acids I; L; M;Amino acid W is similar to amino acids F; Y; andAmino acid Y is similar to amino acids F; H; W.

Conservative amino acid substitutions may occur over the full length ofthe sequence of a polypeptide sequence of a functional protein such asan enzyme. In one embodiment, such mutations are not pertaining thefunctional domains of an enzyme. In one embodiment, conservativemutations are not pertaining the catalytic centers of an enzyme.

Therefore, according to the present description the followingcalculation of percent-similarity applies:

%-similarity=[(identical residues+similar residues)/length of thealignment region which is showing the respective sequence of thisdescription over its complete length]*100.

Especially, variant enzymes comprising conservative mutations which areat least m % similar to the respective parent sequences with m being aninteger between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85,90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 compared to the full-lengthpolypeptide sequence, are expected to have essentially unchanged enzymeproperties. Preferably, variant enzymes with m %-similarity whencompared to a parent enzyme, have enzymatic activity.

Homologous refers to a gene, polypeptide, polynucleotide with a highdegree of similarity, e.g. in position, structure, function orcharacteristic, but not necessarily with a high degree of sequenceidentity.

As used herein, “substantially complementary or substantially matched”means that two nucleic acid sequences have at least about 90% sequenceidentity. Preferably, the two nucleic acid sequences have at least, orat least about, 95%, 96%, 97%, 98%, 99%, or 100% of sequence identity.Alternatively, “substantially complementary or substantially matched”means that two nucleic acid sequences can hybridize under highstringency condition(s).

The term “hybridization” as defined herein is a process whereinsubstantially complementary nucleotide sequences anneal to each other.The hybridization process can occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. The hybridization processcan also occur with one of the complementary nucleic acids immobilizedto a matrix such as magnetic beads, Sepharose beads or any other resin.The hybridization process can furthermore occur with one of thecomplementary nucleic acids immobilized to a solid support such as anitro-cellulose or nylon membrane or immobilized by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids. Hybridization accordingto this description means, that hybridization must occur over completelength of the sequence of the invention. Such hybridization over thecomplete length, as defined herein, means, that when the sequence ofthis invention is fragmented into pieces of 300-500 bases, each fragmentwill hybridize

The term “stringency” refers to the conditions under which ahybridization takes place. The stringency of hybridization is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridization buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthand ph. Medium stringency conditions are when the temperature is 20° C.below Tm, and high stringency conditions are when the temperature is 10°C. below Tm. High stringency hybridization conditions are typically usedfor isolating hybridizing sequences that have high sequence identity tothe target nucleic acid sequence. However, nucleic acids may deviate insequence and still encode a substantially identical polypeptide, due tothe degeneracy of the genetic code. Therefore, medium stringencyhybridization conditions may sometimes be needed to identify suchnucleic acid molecules. The “Tm” is the temperature under defined ionicstrength and pH, at which 50% of the target sequence hybridizes to aperfectly matched probe. The Tm is dependent upon the solutionconditions and the base composition and length of the probe. Forexample, longer sequences hybridize specifically at higher temperatures.The maximum rate of hybridization is obtained from about 16° C. up to32° C. below Tm. The presence of monovalent cations in the hybridizationsolution reduce the electrostatic repulsion between the two nucleic acidstrands thereby promoting hybrid formation; this effect is visible forsodium concentrations of up to 0.4M (for higher concentrations, thiseffect may be ignored). Formamide reduces the melting temperature ofDNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percentformamide, and addition of 50% formamide allows hybridization to beperformed at 30 to 45° C., though the rate of hybridisation will belowered. Base pair mismatches reduce the hybridization rate and thethermal stability of the duplexes. On average and for large probes, theTm decreases about 1° C. per % base mismatch. The Tm may be calculatedusing the following equations, depending on the types of hybrids:

-   -   DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:        267-284, 1984):

T _(m)=81.5° C.+16.6×log[Na ⁺]^(a)+0.41×%[G/C ^(b)]−500×[L^(c)]⁻¹−0.61×% formamide

-   -   DNA-RNA or RNA-RNA hybrids:

T _(m)=79.8+18.5(log₁₀[Na ⁺]^(a)+0.58(% G/C ^(b))+11.8(% G/C^(b))²−820/L ^(c)

-   -   oligo-DNA or oligo-RNAs hybrids:    -   For <20 nucleotides: T_(m)=2 (l_(n))    -   For 20-35 nucleotides: T_(m)=22+1.46 (l_(n))    -   ^(a) or for other monovalent cation, but only accurate in the        0.01-0.4 M range.    -   ^(b) only accurate for % GC in the 30% to 75% range.    -   ^(c) L=length of duplex in base pairs.    -   ^(d) Oligo, oligonucleotide; l_(n), effective length of        primer=2×(no. of G/C)+(no. of A/T).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridization buffer, and treatment with RNase. Fornon-related probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridization and which will either maintain or change the stringencyconditions.

Besides the hybridization conditions, specificity of hybridizationtypically also depends on the function of post-hybridization washes. Toremove background resulting from non-specific hybridization, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridization stringency. A positive hybridizationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridizationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridization conditions for DNAhybrids longer than 50 nucleotides encompass hybridization at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridizationconditions for DNA hybrids longer than 50 nucleotides encompasshybridization at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridizing nucleic acid. Whennucleic acids of known sequence are hybridized, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridization solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate. Another example of highstringency conditions is hybridization at 65° C. in 0.1×SSC comprising0.1 SDS and optionally 5×Denhardt's reagent, 100 μg/ml denatured,fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, followed by thewashing at 65° C. in 0.3×SSC.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

As used herein, a “primer” refers to a nucleic acid molecule that cananneal to a template nucleic acid and serves as a starting point for DNAamplification. The primer can be entirely or partially complementary toa specific region of the template polynucleotide, for example 20nucleotides upstream or downstream from a codon of interest. Anon-complementary nucleotide is defined herein as a mismatch. A mismatchmay be located within the primer or at the either end of the primer.Preferably, a single nucleotide mismatch, more preferably two, and morepreferably, three or more consecutive or not consecutive nucleotidemismatches is (are) located within the primer. The primer can have, forexample, from 5 to 200 nucleotides, preferably, from 20 to 80nucleotides, and more preferably, from 43 to 65 nucleotides. Morepreferably, the primer has 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, or 190 nucleotides. A“forward primer” as defined herein is a primer that is complementary toa minus strand of the template polynucleotide. A “reverse primer” asdefined herein is a primer complementary to a plus strand of thetemplate polynucleotide. Preferably, the forward and reverse primers donot comprise overlapping nucleotide sequences. “Do not compriseoverlapping nucleotide sequences” as defined herein means that a forwardand reverse primer does not anneal to a region of the minus and plusstrands, respectively, of the template polynucleotide in which the plusand minus strands are complimentary to one another. With regard to theprimers annealing to the same strand of the template polynucleotide, “donot comprise overlapping nucleotide sequences” means the primers do notcomprise sequences complementary to the same region of the same strandof the template polynucleotide. As used herein, a “primer set” refers toa combination of a “forward primer” and a corresponding “reverseprimer.”

As used herein, the plus strand equivalent to the sense strand and mayalso be referred to as a coding or non-template strand. This is thestrand that has the same sequence as the mRNA (except it has Ts insteadof Us). The other strand, called the template, minus, or antisensestrand, is complementary to the mRNA.

As described herein, “codon optimization” refers to the design processof altering codons to codons known to increase maximum proteinexpression efficiency. In some alternatives, codon optimization forexpression in a cell is described, wherein codon optimization can beperformed by using algorithms that are known to those skilled in the artso as to create synthetic genetic transcripts optimized for high mRNAand protein yield in a host cell of interest, for example bacterial,fungal, insect, or mammalian cells (including human cells). Codons canbe optimized for protein expression in a bacterial cell, mammalian cell,yeast cell, insect cell, or plant cell, for example. Programs containingalgorithms for codon optimization in human cells are readily available.Such programs can include, for example, OptimumGene™ or GeneGPS®algorithms. Additionally codon optimized sequences can be obtainedcommercially, for example, from Integrated DNA Technologies. In someembodiments, the genes are codon optimized for expression in bacterial,yeast, fungal or insect cells.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction may beelectrophoresed on a gel.

An enzyme is a biological molecule comprising a sequence of amino acids,wherein the enzyme can catalyze a reaction. Enzyme names are known tothose skilled in the art based on the recommendations of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (IUBMB). Enzyme names include: an EC (EnzymeCommission) number, recommended name, alternative names (if any),catalytic activity, and other factors. An enzyme is also known as apolypeptide, a protein, a peptide, an amino acid sequence, or isidentified by a SEQ ID NO. In this disclosure, the alternative names forenzyme can be used interchangeably.

The term “heterologous” (or exogenous or foreign or recombinant)polypeptide is defined herein as:

-   -   (a) a polypeptide that is not native to the host cell. The        protein sequence of such a heterologous polypeptide is a        synthetic, non-naturally occurring, “man made” protein sequence;    -   (b) a polypeptide native to the host cell in which structural        modifications, e.g., deletions, substitutions, and/or        insertions, have been made to alter the native polypeptide; or    -   (c) a polypeptide native to the host cell whose expression is        quantitatively altered or whose expression is directed from a        genomic location different from the native host cell as a result        of manipulation of the DNA of the host cell by recombinant DNA        techniques, e.g., a stronger promoter.

Descriptions b) and c), above, refer to a sequence in its natural formbut not naturally expressed by the cell used for its production. Theproduced polypeptide is therefore more precisely defined as a“recombinantly expressed endogenous polypeptide”, which is not incontradiction to the above definition but reflects the specificsituation that it's not the sequence of a protein being synthetic ormanipulated but the way the polypeptide molecule is produced.

Similarly, the term “heterologous” (or exogenous or foreign orrecombinant) polynucleotide refers:

-   -   (a) to a polynucleotide that is not native to the host cell;    -   (b) a polynucleotide native to the host cell in which structural        modifications, e.g., deletions, substitutions, and/or        insertions, have been made to alter the native polynucleotide;    -   (c) a polynucleotide native to the host cell whose expression is        quantitatively altered as a result of manipulation of the        regulatory elements of the polynucleotide by recombinant DNA        techniques, e.g., a stronger promoter; or    -   (d) a polynucleotide native to the host cell, but integrated not        within its natural genetic environment as a result of genetic        manipulation by recombinant DNA techniques.

With respect to two or more polynucleotide sequences or two or moreamino acid sequences, the term “heterologous” is used to characterizethat the two or more polynucleotide sequences or two or more amino acidsequences do not occur naturally in the specific combination with eachother.

As used herein, “transgenic”, “transgene” or “recombinant” means withregard to, for example, a nucleic acid sequence, an expression cassette,genetic construct or a vector comprising the nucleic acid sequence or anorganism transformed with the nucleic acid sequences, expressioncassettes or vectors, all those constructions brought aboutsynthetically by recombinant or genetechnological methods in whicheither

-   -   (a) the nucleic acid sequences comprising desired genetic        information to be expressed, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence comprising said desired genetic        information, for example a promoter, or    -   (c) both (a) and (b),        are not located in their natural genetic environment or have        been modified by recombinant methods. The natural genetic        environment is understood as meaning the natural genomic or        chromosomal locus in the original organism. A naturally        occurring expression cassette—for example the naturally        occurring combination of the natural promoter of the nucleic        acid sequences with the corresponding nucleic acid sequence        encoding a polypeptide, becomes a transgenic expression cassette        when this expression cassette is modified through human        intervention such as, for example, mutagenic treatment. Suitable        methods are described, for example, in U.S. Pat. No. 5,565,350,        US200405323 and WO 00/15815. Furthermore, a naturally occurring        expression cassette becomes a recombinant expression cassette        when this expression cassette is isolated from its natural        genetic environment and subsequently reintroduced in a genetic        environment that is not the natural genetic environment.

A “synthetic” or “artificial” compound is produced by in vitro chemicalor enzymatic synthesis. It includes, but is not limited to, variantnucleic acids made with optimal codon usage for host organisms, such asa yeast cell host or other expression hosts of choice or variant proteinsequences with amino acid modifications, such as e.g. substitutions,compared to the parent protein sequence—e.g. to optimize properties ofthe polypeptide.

The term “restriction site” refers to a recognition sequence that isnecessary for the manifestation of the action of a restriction enzyme,and includes a site of catalytic cleavage. It is appreciated that a siteof cleavage may or may not be contained within a portion of arestriction site that comprises a low ambiguity sequence (i.e. asequence containing the principal determinant of the frequency ofoccurrence of the restriction site). Thus, in many cases, relevantrestriction sites contain only a low ambiguity sequence with an internalcleavage site (e.g. G/AATTC in the EcoRI site) or an immediatelyadjacent cleavage site (e.g. /CCWGG in the EcoRII site). In other cases,relevant restriction enzymes (e.g. the Eco57I site or CTGAAG(16/14))contain a low ambiguity sequence (e.g. the CTGAAG sequence in the Eco57Isite) with an external cleavage site (e.g. in the N16 portion of theEco57I site). When an enzyme (e.g. a restriction enzyme) is said to“cleave” a polynucleotide, it is understood to mean that the restrictionenzyme catalyzes or facilitates a cleavage of a polynucleotide.

An “ambiguous base requirement” in a restriction site refers to anucleotide base requirement that is not specified to the fullest extent,i.e. that is not a specific base (such as, in a non-limitingexemplification, a specific base selected from A, C, G and T), butrather may be any one of at least two or more bases. Commonly acceptedabbreviations that are used in the art as well as herein to representambiguity in bases include the following: R=G or A; Y=C or T; M=A or C;K=G or T; S=G or C; W=A or T; H=A or C or T; B=G or T or C; V=G or C orA; D=G or A or T; N=A or C or G or T.

A “reference sequence” is a defined sequence used as a basis for asequence comparison; a reference sequence may be a subset of a largersequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the two polynucleotidesand (2) may further comprise a sequence that is divergent between thetwo polynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity.

A “comparison window,” as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, JTeor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, JMol Evol, 1981), by the homology alignment algorithm of Needleman(Needleman and Wuncsch, 1970), by the search of similarity method ofPearson (Pearson and Lipman, 1988), by computerized implementations ofthese algorithms BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by inspection, and the best alignment (i.e.,resulting in the highest percentage of homology over the comparisonwindow) generated by the various methods is selected.

The terms “fragment”, “derivative” and “analog” when referring to areference polypeptide comprise a polypeptide which retains at least onebiological function or activity that is at least essentially same asthat of the reference polypeptide.

The term “functional fragment” refers to any nucleic acid or amino acidsequence which comprises merely a part of the full length nucleic acidor full length amino acid sequence, respectively, but still has the sameor similar activity and/or function. In one embodiment, the fragmentcomprises at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% of theoriginal sequence. In one embodiment, the functional fragment comprisescontiguous nucleic acids or amino acids compared to the original nucleicacid or original amino acid sequence, respectively.

The term “pro-form”, “pro-protein”, or “pro-peptide”, refers to aprotein precursor, which is an inactive or low activity protein (orpeptide) that can be turned into an active or more active form bypost-translational modification, such by cleavage or by addition ofanother peptide or molecule, to produce a mature protein (e.g., to forman enzyme from a pro-enzyme).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as optionally interveningsequences (introns) between individual coding segments (exons).

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor enzyme present in a living animal is not isolated, but the samepolynucleotide or enzyme, separated from some or all of the coexistingmaterials in the natural system, is isolated. Such polynucleotides couldbe part of a vector and/or such polynucleotides or enzymes could be partof a composition, and still be isolated in that such vector orcomposition is not part of its natural environment. As further example,an isolated nucleic acid, e.g., a DNA or RNA molecule, is one that isnot immediately contiguous with the 5′ and 3′ flanking sequences withwhich it normally is immediately contiguous when present in thenaturally occurring genome of the organism from which it is derived.Such polynucleotides could be part of a vector, incorporated into agenome of a cell with an unrelated genetic background (or into thegenome of a cell with an essentially similar genetic background, but ata site different from that at which it naturally occurs), or produced byPCR amplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription, and/or such polynucleotides,polypeptides, or enzymes could be part of a composition, and still beisolated in that such vector or composition is not part of its naturalenvironment.

The term “isolated” means that the DNA is incorporated into a vector,such as a plasmid or viral vector; a nucleic acid that is incorporatedinto the genome of a heterologous cell (or the genome of a homologouscell, but at a non-naturally occurring site); and a nucleic acid thatexists as a separate molecule, e.g., a DNA fragment produced by PCRamplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription.

As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. For example, the purified nucleic acids ofthe present disclosure can be purified from the remainder of the genomicDNA in the organism by at least 10⁴-10⁶ fold. However, the term“purified” also includes nucleic acids which have been purified from theremainder of the genomic DNA or from other sequences in a library orother environment by at least one order of magnitude, typically two orthree orders, and more typically four or five orders of magnitude“Purified” means that the material is in a relatively pure state, e.g.,at least about 90% pure, at least about 95% pure, or at least about 98%or 99% pure. Preferably “purified” means that the material is in a 100%pure state.

The term “operably linked” means that the described components are in arelationship permitting them to function in their intended manner. Forexample, a regulatory sequence operably linked to a coding sequence isligated in such a way that expression of the coding sequence is achievedunder condition compatible with the control sequences. As used herein, apromoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter can transcribethe coding sequence into mRNA.

The term “mutations” is defined as alterations in the genetic code ofnucleic acid sequence or alterations in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions. Amutation may be a change to one or more nucleotides or encoded aminoacid sequences. The mutations may be deletions, insertions orduplications.

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are usedinterchangeably herein and refer to nucleotides, either ribonucleotidesor deoxyribonucleotides or a combination of both, in a polymericunbranched form of any length.

For nucleotide sequences, e.g., consensus sequences, an IUPAC nucleotidenomenclature (Nomenclature Committee of the International Union ofBiochemistry (NC-IUB) (1984). “Nomenclature for Incompletely SpecifiedBases in Nucleic Acid Sequences”.) is used, with the followingnucleotide and nucleotide ambiguity definitions, relevant to thisdescription: A, adenine; C, cytosine; G, guanine; T, thymine; K, guanineor thymine; R, adenine or guanine; W, adenine or thymine; M, adenine orcytosine; Y, cytosine or thymine; D, not a cytosine; N, any nucleotide.

In addition, notation “N(3-5)” means that indicated consensus positionmay have 3 to 5 any (N) nucleotides. For example, a consensus sequence“AWN(4-6)” represents 3 possible variants—with 4, 5, or 6 anynucleotides at the end: AWNNNN, AWNNNNN, AWNNNNNN.

The terms “nucleic acid sequence coding for” or a “DNA coding sequenceof” or a “nucleotide sequence encoding” a particular protein orpolypeptide refer to a DNA sequence which is transcribed and translatedinto a protein or polypeptide when placed under the control ofappropriate regulatory sequences.

The terms “nucleic acid encoding a protein or peptide” or “DNA encodinga protein or peptide” or “polynucleotide encoding a protein or peptide”and other synonymous terms encompasses a polynucleotide which includesonly coding sequence for the protein or peptide as well as apolynucleotide which includes additional coding and/or non-codingsequence.

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are associated. “Regulatoryelements” or “regulatory nucleotide sequences” herein may mean pieces ofnucleic acid which drive expression of a nucleic acid sequence. upontransformation into a host cell or cell organelle had occurred.Regulatory nucleotide sequences may include any nucleotide sequencehaving a function or purpose individually and within a particulararrangement or grouping of other elements or sequences within thearrangement. Examples of regulatory nucleotide sequences include but arenot limited to transcription control elements such as promoters,enhancers, and termination elements. Regulatory nucleotide sequences maybe native (i.e. from the same gene) or foreign (i.e. from a differentgene) to a nucleotide sequence to be expressed.

The term “promoter” typically refers to a nucleic acid control sequencelocated upstream from the transcriptional start of a gene and isinvolved in recognizing and binding of RNA polymerase and otherproteins, thereby directing transcription of an operably linked nucleicacid. “Promoter” herein may further include any nucleic acid sequencecapable of driving transcription of a coding sequence. In particular,the term “promoter” as used herein may refer to a polynucleotidesequence generally described as the 5′ regulator region of a gene,located proximal to the start codon. The transcription of one or morecoding sequence is initiated at the promoter region. The term promotermay also include fragments of a promoter that are functional ininitiating transcription of the gene. Promoter may also be called“transcription start site” (TSS).

Encompassed by the aforementioned terms are further transcriptionalregulatory sequences derived from a classical eukaryotic genomic gene(including the TATA box which is required for accurate transcriptioninitiation, with or without a CCAAT box sequence) and additionalregulatory elements (i.e. upstream activating sequences, enhancers andsilencers) which alter gene expression in response to developmentaland/or external stimuli, or in a tissue-specific manner.

For example, enhancers as known in the art and as used herein arenormally short DNA segments (e.g. 50-1500 bp) which may be bound byproteins such as transcription factors to increase the likelihood thattranscription of a coding sequence will occur.

Also included within the term is a transcriptional regulatory sequenceof a classical prokaryotic gene, in which case it may include a −35 boxsequence and/or −10 box transcriptional regulatory sequences. The term“regulatory element” also encompasses a synthetic fusion molecule orderivative that confers, activates or enhances expression of a nucleicacid molecule in a cell, tissue or organ. A promoter can be modified byone or more nucleotide substitution(s), insertion(s) and/or deletion(s)without interfering with functionality or activity, but it is alsopossible to increase the activity by modification of its sequence.

Further elements may be “transcription termination elements” whichinclude pieces of nucleic acid sequences marking the end of a gene andmediating the transcriptional termination by providing signals withinmRNA that initiates the release of the mRNA from the transcriptionalcomplex. Transcriptional termination in prokaryotes usually is initiatedby Rho-dependent or Rho-independent terminators. In eukaryotestranscription termination usually occurs through recognition oftermination by proteins associated with RNA polymerase II.

An “oligonucleotide” (or synonymously an “oligo”) refers to either asingle stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands which may be chemically synthesized. Suchsynthetic oligonucleotides may or may not have a 5′ phosphate. Thosethat do not will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

Any source of nucleic acid, in purified form can be utilized as thestarting nucleic acid (also defined as “a template polynucleotide”).Thus, the process may employ DNA or RNA including messenger RNA, whichDNA or RNA can be single-stranded, and preferably double stranded. Inaddition, a DNA-RNA hybrid which contains one strand of each may beutilized. The nucleic acid sequence may be of various lengths dependingon the size of the nucleic acid sequence to be mutated. Preferably thespecific nucleic acid sequence is from 50 to 50000 base pairs, and morepreferably from 50-11000 base pairs.

Standard convention (5′ to 3′) is used herein to describe the sequenceof double-stranded polynucleotides.

All methods and materials similar or equivalent to those describedherein can be used in the practice or testing of methods andcompositions disclosed herein, with suitable methods and materials beingdescribed herein. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. Further, the materials, methods, and examples are illustrativeonly and are not intended to be limiting, unless otherwise specified.

Nuclease Polypeptides

Disclosed herein are polypeptides having nuclease activity. In someembodiments, the polypeptide is an isolated, synthetic, or recombinantpolypeptide comprising an amino acid sequence having at least 80%, 85%,90%, 95%, 98%, 99%, or more sequence identity to SEQ ID NO: 1, where thepolypeptide has nuclease activity. The polypeptide can, for example, has80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or arange between any two of these values, sequence identity to SEQ IDNO: 1. As described herein, a polypeptide having the amino acid sequenceof SEQ ID NO: 1 exhibits nuclease activity. The first 21 amino acids onthe N-terminus of SEQ ID NO: 1 is the signal sequence, and the remainingamino acids form the mature polypeptide. The coding nucleic acidsequence of SEQ ID NO: 1 is disclosed herein as SEQ ID NO: 2. The aminoacid mutations are described herein relative to the corresponding aminoacid position in SEQ ID NO: 1. For example, an amino acid substitutionfrom E to L at position 230 of SEQ ID NO: 1 is described herein asE230L.

In some embodiments, the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, V226K, E230R,G263A, G119N, V226K, G263A, N127S, P84V, D83E, D28G, V45T, M262V, Al90K, P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E,P72N, P84L, S74N, T82R, G75R, Q141R, and D107N. In some embodiments, thepolypeptide comprises one or more mutations of L114F, E230M, D107N,Q141R, G119N, S74N, T82R, and G75R. In some embodiments, the polypeptidecomprises a combination of mutations selected from the group consistingof: (a) T82R, L114F, and G119N; (b) G75R, T82R, L114F, and G119N; (c)S74N, G75R, T82R, G119N, and Q141R; (d) S74N, T82R, L114F, and G119N;(e) S74N, T82R, L114F, G119N, and Q141R; (f) S74N, T82R, G119N, andQ141R; (g) S74N, L114F, and Q141R; (h) S74N, L114F, G119N, and Q141R;(i) S74N, G75R, T82R, L114F, and Q141R; (j) S74N, G75R, T82R, L114F,G119N, and Q141R; (k) S74N, G75R, T82R, and Q141R; (1) S74N, G75R,L114F, and Q141R; (m) S74N, G75R, and Q141R; (n) T82R, D107N, and G119N;(o) S74N, G75R, T82R, D107N, and Q141R; (p) S74N, G75R, G119N, andQ141R; (q) T82R, D107N, and L114F; (r) G75R, T82R, D107N, and L114F; (s)G75R, D107N, and L114F; (t) T82R, D107N, L114F, and G119N; and (u) S74N,G75R, L114F, G119N, and Q141R. In some embodiments, the polypeptidecomprises a mutation or a combination of mutations selected from thegroup consisting of:

-   -   (T82R, L114F);    -   (T82R, L114F, G119N);    -   (T82R)    -   (T82R, G119N);    -   (L114F);    -   (L114F, G119N);    -   (G119N);    -   (G75R, T82R, L114F, G119N);    -   (G75R, T82R, L114F, G119N);    -   (G75R, T82R);    -   (G75R, L114F);    -   (G75R, L114F, G119N);    -   (S74N, G75R, T82R, G119N, Q141R);    -   (S74N, T82R, L114F, G119N);    -   (S74N, T82R, L114F, G119N, Q141R);    -   (S74N, T82R, Q141R);    -   (S74N, T82R, G119N, Q141R);    -   (S74N, L114F, Q141R);    -   (S74N, L114F, G119N, Q141R);    -   (S74N, Q141R);    -   (S74N, G75R, T82R, L114F, Q141R);    -   (S74N, G75R, T82R, L114F, G119N, Q141R);    -   (S74N, G75R, T82R, Q141R);    -   (S74N, G75R, L114F, Q141R);    -   (S74N, G75R, Q141R);    -   (T82R, D107N);    -   (G75R, T82R, D107N);    -   (D107N);    -   (G75R, T82R, G119N);    -   (T82R, D107N, G119N);    -   (G75R, T82R, D107N, G119N);    -   (D107N, G119N);    -   (G75R, D107N, G119N);    -   (S74N, T82R, D107N, Q141R);    -   (S74N, G75R, T82R, D107N, Q141R);    -   (S74N, G75R, D107N, Q141R);    -   (S74N, G119N, Q141R);    -   (S74N, G75R, G119N, Q141R);    -   (S74N, T82R, D107N, G119N, Q141R);    -   (S74N, G75R, T82R, D107N, G119N, Q141R);    -   (S74N, D107N, G119N, Q141R);    -   (S74N, G75R, D107N, G119N, Q141R);    -   (T82R, D107N, L1140;    -   (G75R, T82R, D107N, L1140;    -   (D107N, L1140;    -   (G75R, D107N, L1140;    -   (G75R, D107N);    -   (T82R, D107N, L114F, G119N);    -   (D107N, L114F, G119N);    -   (G75R, D107N, L114F, G119N);    -   (S74N, T82R, D107N, L114F, Q1414    -   (S74N, G75R, T82R, D107N, L114F, Q1414    -   (S74N, D107N, L114F, Q1414    -   (S74N, G75R, D107N, L114F, Q1414    -   (S74N, D107N, Q1414    -   (S74N, G75R, L114F, G119N, Q1414    -   (S74N, T82R, D107N, L114F, G119N, Q141R);    -   (S74N, G75R, T82R, D107N, L114F, G119N, Q141R);    -   (S74N, D107N, L114F, G119N, Q141R);    -   (S74N, G75R, D107N, L114F, G119N, Q141R); and    -   (G75R, T82R, D107N, L114F, G119N).        As used herein, a combination of amino acid mutations can be        described as “Mutation1, Mutation2, and Mutation3”, for example        one non-limiting exemplary combination of mutations is “T82R,        D107N, and G119N”, or be described as (Mutation 1, Mutation 2,        Mutation 3), for example one non-limiting exemplary combination        of mutations is (T82R, D107N, G119N).

In some embodiments, the polypeptide is any one of the nuclease variantsdisclosed herein, where the polypeptide has nuclease activity.

In some embodiments, the nuclease polypeptides are thermostable,thermotolerant, or both. For example, the nuclease polypeptide can bemore thermostable, thermotolerant, or both than the nuclease having thesequence of SEQ ID NO: 1 and/or its parent nuclease. In someembodiments, the nuclease activity of the polypeptide is at least 1%,2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or morehigher than that of the nuclease having the sequence of SEQ ID NO: 1 ata given temperature, for example at a temperature between 10° C. and 70°C., or a temperature between 37° C. and 60° C., or a temperature between40° C. and 55° C., or a temperature of 37° C. In some embodiments, thenuclease activity of the polypeptide is 1%, 2%, 3%, 4%, 5%, 7%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, or a range betweentwo of these values, higher than that of the nuclease having thesequence of SEQ ID NO: 1 at a given temperature, for example at atemperature between 10° C. and 70° C., at a temperature between 37° C.and 60° C., or temperature between 40° C. and 55° C., or a temperatureof 37° C. In some embodiments, the nuclease activity of the polypeptideis at least 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, or more higher than that of the nuclease having the sequenceof SEQ ID NO: 1 at a given pH, for example at pH 4 to pH 11, or at pH 6to pH 7.5, or at pH 6 to pH 7, or at pH 6.5 to pH 7, or at pH 6.5.

In some embodiments, the nuclease activity ratio 54° C./37° C. of thepolypeptide is at least 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, or more higher than that of the nuclease having thesequence of SEQ ID NO: 1. In some embodiments, the nuclease activityratio 54° C./37° C. of the polypeptide is 1%, 2%, 3%, 4%, 5%, 7%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, 100%, or a range betweentwo of these values, higher than that of the nuclease having thesequence of SEQ ID NO: 1.

The optimal temperature of the polypeptide can be different (for examplehigher or lower) than that of the nuclease having the sequence of SEQ IDNO: 1 or its parent nuclease. For example, the optimal temperature ofthe polypeptide can be 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C.,8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 20° C.,25° C., or a range between any two of these values, higher than theoptimal temperature of the nuclease having the sequence of SEQ ID NO: 1or its parent nuclease. In some embodiments, the optimal temperature ofthe polypeptide is at least 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7°C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 20°C., 25° C., or more, higher than the optimal temperature of the nucleasehaving the sequence of SEQ ID NO: 1 or its parent nuclease. In someembodiments, the optimal temperature of the polypeptide is, or is about,10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C.,55° C., 60° C., 65° C., 70° C., 75° C., or a range between any two ofthese values. In some embodiments, the optimal temperature of thepolypeptide is between 10° C. and 70° C. In some embodiments, theoptimal temperature of the polypeptide is between 37° C. and 60° C.

The optimal pH of the polypeptide can be different (for example lower orhigher) than that of the nuclease having the sequence of SEQ ID NO: 1and/or its parent nuclease. For example, the difference between theoptimal pH of the polypeptide and the optimal pH of the nuclease havingthe sequence of SEQ ID NO: 1 and/or its parent nuclease can be pH 0.05,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7,0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2, 2.5, 3, or more, or a range between any two of these values. Insome embodiments, the optimal pH of the polypeptide is higher than theoptimal pH of the nuclease having the sequence of SEQ ID NO: 1 and/orits parent nuclease. In some embodiments, the optimal pH of thepolypeptide is lower than the optimal pH of the nuclease of SEQ ID NO: 1and/or its parent nuclease. In some embodiments, the difference betweenthe optimal pH of the polypeptide and the optimal pH of the nucleasehaving the sequence of SEQ ID NO: 1 and/or its parent nuclease is atleast, or at most, pH 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45,0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3. The optimal pH of thepolypeptide can be, for example, 4, 4.5, 5, 5.5, 6, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.4, or 11, or arange between any two of these values. The optimal pH of the polypeptidecan be at least, or be at most, pH 4, 4.5, 5, 5.5, 6, 6.1, 6.2, 6.3,6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.4, or 11.In some embodiments, the optimal pH of the polypeptide is between pH 4to pH 11, pH 6 to pH 7, for example pH 6.5.

During the fermentation process, DNA from a production host cancomplicate many aspects of the protein recovery process. Currently,DNAse has been added in order to remove the DNA from final product,which often requires addition of costly materials from other sources.The nucleases disclosed herein can be expressed, in some embodiments, inthe same production host as product of interest, which eliminates theneeds to add external DNAse so that can reduce the overall cost ofprocessing to final product.

The nuclease polypeptides disclosed herein can have one or more signalsequences. In some embodiments, at least one of the one or more signalsequences is heterologous to the nuclease polypeptide it is comprisedin. In some embodiments, the nuclease polypeptides disclosed herein donot contain any signal sequences.

Also disclosed herein are antibody or binding fragment thereof (e.g.,isolated or purified antibody or binding fragment thereof) whichspecifically binds to an isolated, synthetic, or recombinant polypeptidecomprising an amino acid sequence having at least 80%, 85%, 90%, 95%,98%, 99%, or more sequence identity to SEQ ID NO: 1, where thepolypeptide has nuclease activity. The polypeptide can, for example, has80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or arange between any two of these values, sequence identity to SEQ IDNO: 1. In some embodiments, the polypeptide comprises one or moremutations of E230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H,V226K, E230R, G263A, G119N, G263A, N127S, P84V, D83E, D28G, V45T, M262V,A190K, P84N, I44R, G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E,P72N, P84L, S74N, T82R, G75R, G119N, Q141R, and D107N. In someembodiments, the polypeptide comprises a mutation of L114F, E230M,D107N, Q141R, G119N, S74N, T82R, or G75R. In some embodiments, thepolypeptide comprises a combination of mutations selected from the groupconsisting of: (a) T82R, L114F, and G119N; (b) G75R, T82R, L114F, andG119N; (c) S74N, G75R, T82R, G119N, and Q141R; (d) S74N, T82R, L114F,and G119N; (e) S74N, T82R, L114F, G119N, and Q141R; (f) S74N, T82R,G119N, and Q141R; (g) S74N, L114F, and Q141R; (h) S74N, L114F, G119N,and Q141R; (i) S74N, G75R, T82R, L114F, and Q141R; (j) S74N, G75R, T82R,L114F, G119N, and Q141R; (k) S74N, G75R, T82R, and Q141R; (1) S74N,G75R, L114F, and Q141R; (m) S74N, G75R, and Q141R; (n) T82R, D107N, andG119N; (o) S74N, G75R, T82R, D107N, and Q141R; (p) S74N, G75R, G119N,and Q141R; (q) T82R, D107N, and L114F; (r) G75R, T82R, D107N, and L114F;(s) G75R, D107N, and L114F; (t) T82R, D107N, L114F, and G119N; and (u)S74N, G75R, L114F, G119N, and Q141R.

Variants of the nucleases disclosed herein can comprise one or more ofsubstitutions, deletions, and insertions at one or more of the aminoacid positions of the nucleases. In some embodiments, the number ofamino acid substitutions, deletions and/or insertions introduced intothe parent nuclease (for example the nuclease having the sequence of SEQID NO: 1) is not more than 30, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, or 29. The amino acid changes can be of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions (for example 1-20 amino acids); small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain. In some embodiments, the amino acid changes to thenucleases can alter one or more physico-chemical properties of theparent nucleases. For example, the amino acid changes may alter (e.g.,improve or decrease) one or more of the properties of the nucleasepolypeptides, including but not limited to, thermal stability, substratespecificity, pH optimum, temperature optimum, and the like as comparedto the parent nuclease(s).

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Non-limitingexemplary amino acid substitutions include Ala to Ser, Val to Ile, Aspto Glu, Thr to Ser, Ala to Gly, Ala to Thr, Ser to Asn, Ala to Val, Serto Gly, Tyr to Phe, Ala to Pro, Lys to Arg, Asp to Asn, Leu to Ile, Leuto Val, Ala to Glu, and Asp to Gly.

Amino acid substitutions, deletions, and/or insertions can be made andtested using methods known in the art for protein/DNA engineering,including but not limited to mutagenesis, recombination, and/orshuffling, followed by a relevant screening procedure, such as thosedisclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowieand Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;or WO 95/22625. Other methods that can be used include error-prone PCR,phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837;U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis(Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).In some embodiments, mutagenesis/shuffling methods can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides expressed by host cells. MutagenizedDNA molecules that encode active polypeptides can be recovered from thehost cells and rapidly sequenced using standard methods in the art.These methods allow the rapid determination of the importance ofindividual amino acid residues in a polypeptide.

In some embodiments, the nuclease polypeptides can tolerate higher orlower pH than other nucleases, for example the nuclease having thesequence of SEQ ID NO: 1. For example, the nuclease polypeptides canretain a nuclease activity (for example, at least 40%, 50%, 60%, 70%,80%, 90%, 95%, or 98% of its nuclease activity) at, or at about, pH 3.0,pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5,pH 8.0, pH 8.5, pH 9.0, pH 9.5, pH 10.0, pH 10.5, pH 11.0, pH 11.5, pH12.0, or a range between any two of these values. For example, thenuclease polypeptides retain a nuclease activity (for example, at least40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% of its nuclease activity)above, or below, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0,pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0, pH 9.5, pH 10.0, pH10.5, pH 11.0, pH 11.5, pH 12.0, or a range between any two of thesevalues. In some embodiments, these pH tolerant nuclease polypeptides arealso thermostable. For example, the thermostable nuclease polypeptidescan retain a nuclease activity (for example, at least 40%, 50%, 60%,70%, 80%, 90%, 95%, or 98% of its nuclease activity) at a temperature at10° C., 15° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 41° C.,42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C.,51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 56° C., 57° C., 58° C.,59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 70° C., or arange between any two of these values.

The nuclease polypeptides disclosed herein can have the same ordifferent substrate specificity as compared to the nuclease having thesequence of SEQ ID NO: 1 or its respective parent nuclease. For example,the nuclease polypeptides can have substantially the same substratespecificity as compared to the nuclease having the sequence of SEQ IDNO: 1 or its respective parent nuclease. In some embodiments, thenuclease polypeptides have about 50%, 60%, 70%, 80%, 90%, 95%, 98%, or arange between any two of these values, substrate specificity as comparedto the nuclease having the sequence of SEQ ID NO: 1 or its respectiveparent nuclease. Also disclosed herein is a composition that comprisesone or more of the nuclease polypeptides disclosed herein.

Also provided herein are immobilized nuclease polypeptides, wherein theimmobilized polypeptide comprises one of the nuclease polypeptidesdisclosed herein. In some embodiments, the polypeptide can beimmobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass,a microelectrode, a graphitic particle, a bead, a gel, a plate, anarray, a capillary tube, or a combination thereof.

Production of Nuclease Polypeptides and Variants Thereof

Provided herein are methods for modifying and making variants ofnuclease polynucleotides disclosed herein. Some embodiments providesynthetic or recombinant nucleic acid that encodes one or more of thepolypeptides disclosed herein, and vectors (for example expressionvectors) comprising the nucleic acid. Non-limiting examples of themethod include synthetic ligation reassembly, random mutagenesis,targeted mutagenesis, optimized directed evolution system and/orsaturation mutagenesis such as gene site saturation mutagenesis (GSSM),and any combination thereof. As used herein, the terms “saturationmutagenesis,” “Gene Site Saturation Mutagenesis” and “GSSM” are usedinterchangeably and refers to a method that uses degenerateoligonucleotide primers to introduce point mutations into apolynucleotide. The term “optimized directed evolution system” or“optimized directed evolution” includes a method for reassemblingfragments of related nucleic acid sequences, such as related genes, andexplained in detail, below. The term “synthetic ligation reassembly” or“SLR” includes a method of ligating oligonucleotide fragments in anon-stochastic fashion, and explained in detail, below. The term“variant” refers to polynucleotides or polypeptides in accordance withthe description modified at one or more base pairs, codons, introns,exons, or amino acid residues (respectively) yet still retain thebiological activity of a nuclease. Variants can be produced by methodssuch as by error-prone PCR, shuffling, site-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis(phage-assisted continuous evolution, in vivo continuous evolution),cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

In addition, as described in U.S. Pat. No. 9,476,078 (the content ofwhich is hereby expressly incorporated by reference in its entirety),the tailored multi-sire combinatorial assembly (“TMCA”) method cangenerate a specific gene variant comprising multiple changes or acombinatorial gene library efficiently and quickly; requires minimumcost and effort; and can be tailored to make biased combinatoriallibrary according to the “needs.” The TMCA method can be performedwithout employing a ligation step and, therefore, simplifies the processof generating multiple mutations. The “needs” of a particular libraryvary by experiments. Potential mutation sites—the “needs”—for example,may be either 1) rationally designed amino acid changes or 2) individualamino acids alterations empirically determined to produce a desiredeffect on an enzyme (determined by GSSM and screening efforts). Eachlibrary is created with a specific number of potential mutation sites.It may be preferable to create a library biased towards progeny witheither more or less mutations at the potential mutation sites. Likewise,it may be preferable to create a library in which a bias exists towardsor against a particular mutation or mutation site. Various nucleasepolypeptides disclosed herein were generated using the TMCA method.

Cloning vehicles comprising an expression cassette (such as a vector)can be used herein to express one or more of the nuclease polypeptidesdisclosed herein. The term “vector” as used herein encompasses any kindof cloning vehicles, such as but not limited to plasmids, phagemids,viral vectors (e.g., phages), bacteriophage, baculoviruses, cosmids,fosmids, artificial chromosomes, or and any other vectors specific forspecific hosts of interest. Low copy number or high copy number vectorsare also included. Foreign polynucleotide sequences usually comprise acoding sequence which may be referred to herein as “gene of interest”.The gene of interest may comprise introns and exons, depending on thekind of origin or destination of host cell. The cloning vehicle can be aviral vector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage, an artificial chromosome, or a combination thereof. Theviral vector can comprise an adenovirus vector, a retroviral vector oran adeno-associated viral vector. The cloning vehicle can comprise abacterial artificial chromosome (BAC), a plasmid, a bacteriophageP1-derived vector (PAC), a yeast artificial chromosome (YAC), or amammalian artificial chromosome (MAC). In some embodiments, thepolynucleotide sequence encoding one or more nuclease polypeptides isintegrated into a chromosome of the host cell in which thepolynucleotide sequence is present, and thus the polynucleotide is apart of a chromosomal of the host cell. The host cell can be, forexample, a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, an insect cell, or a plant cell. In some embodiments, thepolynucleotide sequence encoding one or more nuclease polypeptides isnot located in the chromosome of the host cell.

Also provided herein are transformed host cells comprising nucleic acidsor expression cassettes (such as vectors) or cloning vehicles comprisinga nucleic acid sequence that encodes one or more of the nucleasepolypeptides disclosed herein. Some embodiments provide a method forproducing a recombinant polypeptide having nuclease activity, where themethod comprises expressing a polynucleotide encoding one or morenuclease polypeptides disclosed herein under conditions that allowexpression of at least one of the one or more nuclease polypeptides,thereby producing the recombinant polypeptide having nuclease activity.In some embodiments, the polynucleotide encoding one or more nucleasepolypeptides disclosed herein is operably linked to a promoter. In someembodiments, the polypeptide is present in an expression vector. In someembodiments, the polynucleotide is present in the host cell to allowexpression of the polypeptide. In some embodiments, the polynucleotideis present in a chromosome of the host cell to allow expression of thepolypeptide. In some embodiments, the transformed host cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insectcell or a plant cell. In some embodiments, the transformed host cell isa cell from Pichia pastoris (Komagataella pastoris), Bacillus subtilis,Pseudomonas fluorescens, Myceliopthora thermophile fungus, Tricodermeareesei, Escherichia coli, Bacillus licheniformis, Aspergillus niger,Schizosaccharomyces pombe, or. Sacaramyces cerevisiae

Non-limiting examples of expression vectors include viral particles,baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterialartificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul poxvirus, pseudorabies and derivatives of SV40), PI-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors specific for specific hosts of interest (such as bacillus,aspergillus and yeast). Nuclease-encoding DNA disclosed herein can beincluded in any one of a variety of expression vectors for expressing anuclease polypeptide. Such vectors include chromosomal, nonchromosomaland synthetic DNA sequences. Many suitable vectors are known to those ofskill in the art, and are commercially available, for example,bacterial: pQE vectors (Qiagen), pBluescript plasmids, pNH vectors,(lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia); and eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG,pSVLSV40 (Pharmacia). Depending on the desired use, low copy number orhigh copy number vectors can be used.

Codon optimization can be used to achieve high levels of proteinexpression in host cells. In some embodiments, codons in a nucleic acidencoding one or more of the nuclease polypeptides disclosed herein canbe optimized to increase or decrease its expression in a host cell. Forexample, one or more of non-preferred or less preferred codons in thenucleic acid encoding the nuclease polypeptides can be replaced with oneor more “preferred codons” encoding the same amino acid for a host cellof interest. As used herein, a “preferred codon” is a codonover-represented in coding sequences in genes in a host cell, and a“non-preferred or less preferred codon” is a codon under-represented incoding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors in accordance with the present disclosure could be a eukaryoticcell or a prokaryotic cell, and could include bacteria, yeast, fungi,plant cells, insect cells and mammalian cells; and provides methods foroptimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli; gram positive bacteria, such as Streptomyces sp., Lactobacillusgasseri, Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis,and Bacillus cereus. Exemplary host cells also include eukaryoticorganisms, such as various yeast, such as Saccharomyces sp., includingSaccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. In someembodiments, the host cell is a cell from an organism selected from thegroup consisting of Pichia pastoris (Komagataella pastoris), Bacillussubtilis, Pseudomonas fluorescens, Myceliopthora thermophile fungus,Tricodermea reesei, Escherichia coli, Bacillus licheniformis,Aspergillus niger, Schizosaccharomyces pombe, and s. Sacaramycescerevisiae. The nucleic acid encoding the nuclease polypeptide disclosedherein can be located on the genome of the host cell, for example be apart of a chromosome of the host cell. In some embodiments, the nucleicacid encoding the nuclease polypeptide is located on an expressionvector separate from the genome of the host cell. Methods of producing anuclease polypeptide disclosed herein can, in some embodiments,comprise, expressing a nucleic acid encoding the nuclease polypeptideunder conditions that allow expression of the nuclease polypeptide,thereby producing the nuclease polypeptide. In some embodiments, thenucleic acid encoding the nuclease polypeptide is operably linked to aninducible promoter, for example a promoter inducible by changes intemperature and/or pH, and/or by the presence, absence or change inamount/concentration of a compound (e.g., Isopropylβ-D-1-thiogalactopyranoside (IPTG), arabinose, tetracycline, steroids,and metal).

The description also includes nucleic acids and polypeptides optimizedfor expression in these organisms and species.

Use of Nuclease Polypeptides

Some embodiments provide methods for degrading a polynucleotide usingone or more of the nuclease polypeptides disclosed herein. In someembodiments, the method comprises contacting one or more polynucleotidemolecules with one or more of the nuclease polypeptides disclosedherein, thereby degrading the polynucleotide molecules. Thepolynucleotide molecules can comprise DNA (e.g., single- ordouble-stranded DNA), RNA (e.g., single- or double-stranded DNA), or anycombination thereof. The contacting can occur at various pH values, forexample pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, pH 11, or a rangebetween any two of these values. The contacting can also occur atvarious temperatures, for example, 10° C., 15° C., 20° C., 25° C., 30°C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., or arange between any two of these values. The one or more polynucleotidemolecules can be comprised in various objects, for example a compositionfor washing (e.g., textile, clothing, cotton, fabric, or any combinationthereof), or an aqueous solution (e.g., a reaction mixture).

Nucleases have been used in a wide range of applications, for examplepharmaceutical, agricultural and industrial applications. Providedherein are compositions and kits that comprise one or more nucleasepolypeptides disclosed herein. The composition can be an enzymecomposition, a detergent composition, a detergent additive, a food, afood supplement, a feed supplement, a feed, a pharmaceuticalcomposition, a fermentation product, a fermentation intermediate, afermentation downstream reaction mixture, a reaction mixture, or acombination thereof. In some embodiments, the reaction mixture is forprotein expression or purification. The enzyme composition can, forexample, comprise one or more of the nuclease polypeptide disclosedherein and a storage buffer. In some embodiments, the storage buffercomprises a pH buffering system (e.g., Tris-HCl) providing a pH of about6.0-9.0, for example pH 6.0-7.0 or about pH 6.5. The storage buffer can,for example, include a stabilizing agent such as glycerol. In someembodiments, the storage buffer includes glycerol at a concentration ofat least about 5%, 10%, 20%, 30%, 40%, 50%, or more. In someembodiments, the storage buffer includes glycerol at a concentration ofabout 30-70%, for example 40-60%.

Also disclosed herein is a method for degrading polynucleotides duringexpression or production of a protein of interest. The method, in someembodiments, comprises culturing a host cell, wherein the host cellcomprises a nucleic acid encoding a protein of interest; and expressingone or more of the nuclease polypeptides disclosed herein underconditions that allow degradation of polynucleotides by at least one ofthe one or more nuclease polypeptides. The polynucleotide molecules cancomprise DNA, RNA, or any combination thereof. The polynucleotidemolecules can comprise DNAs or RNAs (or fragments thereof) from the hostcell. In some embodiments, the protein of interest is not a nuclease. Insome embodiments, the protein of interest is not any one of the one ormore nuclease polypeptides. The one or more nuclease polypeptides can beexpressed from, for example, one or more expression vectors present inthe host cell, or a nucleic acid sequence in a chromosome of the hostcell. In some embodiments, at least one of the one or more nucleasepolypeptides is not expressed by cells that express the protein ofinterest. In some embodiments, the one or more nuclease polypeptides areexpressed by cells that do not express the protein of interest. Theexpression of the protein of interest and/or the expression of thenuclease polypeptide can be inducible. For example, the coding sequenceof the protein of interest, the coding sequence of the nucleasepolypeptide, or both, can be operably linked with an inducible promoter.The inducible promoter can be, for example, induced by the presence,absence, and/or change in amount of one or more chemical or biologicalcompounds, change in pH, temperature, osmolarity, ionicstrength/concentration, or any combination thereof. Some embodimentsprovide a host cell comprising a nucleic acid encoding a protein ofinterest and a nucleic acid encoding one or more of the nucleasepolypeptides disclosed herein.

The compositions can be formulated in a variety of forms, such astablets, gels, pills, implants, liquids, sprays, films, micelles,powders, food, feed pellets, a type of encapsulated form, or acombination thereof.

In some embodiments, the nuclease polypeptides disclosed herein can beused alone, or in combination with one or more additional enzymes in adetergent application. Some embodiments provide detergent compositionscomprising the nucleases and their variants disclosed herein. In someembodiments, the detergent composition can further comprise one or moreadditional enzymes, or one or more additional components, or anycombination thereof. The detergent composition can be, for example, ahand or machine laundry detergent composition. The one or moreadditional components include but not limited to, one or more laundryadditive compositions suitable for pre-treatment of stained fabrics anda rinse added fabric softener composition. The detergent composition canbe formulated, for example, for use in general household hard surfacecleaning operations, or for hand or machine dishwashing operations.Non-limiting examples of the additional components include surfactants(e.g., anionic surfactants, cationic surfactants, non-ionic surfactants,semi-polar surfactants, zwitterionic surfactants, or a mixture thereof),builders, co-builders, bleach systems, polymers, fabric hueing agents,anti-foaming agents, soil release polymers, anti-redeposition agents,hydrotropes, wetting agents, thickening agents, buffer(s) for pHcontrol, stabilizers, perfume, colorants, fillers and the like, or anycombination thereof. The builder and/or co-builder can be, for example,a chelating agent that forms water-soluble complexes with Ca and Mg.

Some embodiments provide methods of using the nucleases and theirvariants in a detergent application. For example, the method cancomprise (a) contacting a textile to a detergent composition comprisingone or more of the nucleases and their variants disclosed herein, and(b) performing at least one wash cycle to wash the textile. The methodcan, for example, further comprise rinsing the washed textile. In someembodiments, the detergent composition further comprises one or moreadditional enzymes. In some embodiments, the textile is an item made ofcotton or a synthetic material, for example a piece of sportswear, aT-shirt, or a piece of clothing which is exposed to sweat when used. Thetextile can also be bedding, bed linen or towels.

As disclosed herein, the one or more additional enzymes can include, butare not limited to, one or more of nucleases, proteases, lipases,cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases,arabinases, galactanases, xylanases, oxidases (e.g., laccases andperoxidases), and deoxyribonucleases (DNases). The detergent compositioncan, for example, be used in a washing method for textile. In someembodiments, the detergent composition can comprise one or more of thefollowing anionic surfactants: linear alkylbenzenesulfonates (LAS),isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES), secondary alkanesulfonates(SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acidglycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe orSES), methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid,dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives ofamino acids, diesters and monoesters of sulfo-succinic acid or soap.

Also provided are pharmaceutically acceptable prodrugs of thepharmaceutical compositions, and treatment methods employing suchpharmaceutically acceptable prodrugs. The term “prodrug” means aprecursor of a designated compound that, following administration to asubject, yields the compound in vivo via a chemical or physiologicalprocess such as solvolysis or enzymatic cleavage, or under physiologicalconditions (e.g., a prodrug on being brought to physiological pH isconverted to the agent). A “pharmaceutically acceptable prodrug” is aprodrug that is non-toxic, biologically tolerable, and otherwisebiologically suitable for administration to the subject. Illustrativeprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in Bundgaard, Design of Prodrugs(Elsevier Press, 1985).

Also provided are pharmaceutically active metabolites of thepharmaceutical compositions, and uses of such metabolites in the methodsof the description. A “pharmaceutically active metabolite” means apharmacologically active product of metabolism in the body of a compoundor salt thereof. Prodrugs and active metabolites of a compound may bedetermined using routine techniques known or available in the art. See,e.g., Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al.,J Phann. Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34,220-230; Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design ofProdrugs (Elsevier Press, 1985); and Larsen, Design and Application ofProdrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds.,Harwood Academic Publishers, 1991).

Any suitable formulation of the compounds described herein can beprepared. See, generally, Remington's Pharmaceutical Sciences, (2000)Hoover, J. E. editor, 20th edition, Lippincott Williams and WilkinsPublishing Company, Easton, Pa., pages 780-857. A formulation isselected to be suitable for an appropriate route of administration. Someroutes of administration are oral, parenteral, by inhalation, topical,rectal, nasal, buccal, vaginal, via an implanted reservoir, or otherdrug administration methods. In cases where compounds are sufficientlybasic or acidic to form stable nontoxic acid or base salts,administration of the compounds as salts may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids that form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceuticallyacceptable salts are obtained using standard procedures well known inthe art, for example, by a sufficiently basic compound such as an aminewith a suitable acid, affording a physiologically acceptable anion.Alkali metal (e.g., sodium, potassium or lithium) or alkaline earthmetal (e.g., calcium) salts of carboxylic acids also are made.

In some embodiments, the composition comprising one or more nucleasepolypeptides disclosed herein is a pharmaceutical composition or ananti-biofouling composition for disrupting a biofilm, for preventionbiofilm formation, or both. In some embodiments, the pharmaceuticalcomposition is used for treating dental plaque; dental caries;periodontitis; native valve endocarditis; chronic bacterial prostatitis;otitis media; infections associated with medical devices such asartificial heart valves, artificial pacemakers, contact lenses,prosthetic joints, sutures, catheters, and arteriovenous shunts;infections associated with wounds, lacerations, sores and mucosallesions such as ulcers; infections of the mouth, oropharynx, nasopharynxand laryngeal pharynx; infections of the outer ear; infections of theeye; infections of the stomach, small and large intestines; infectionsof the urethra and vagina; infections of the skin; intra-nasalinfections, such as infections of the sinus; or a combination thereof.In some embodiments, the composition comprising one or more nucleasepolypeptides disclosed herein is a pharmaceutical composition for oralcare, treating wound, or both. In some embodiments, the method for usingthe composition comprises contacting the composition with a biofilm. Insome embodiments, the method for using the composition comprisescontacting the composition with a wound, a laceration, a sore, a mucosallesion, or any combination thereof. In some embodiments, the method forusing the composition comprises contacting the composition with amedical device. In some embodiments, the method for using thecomposition comprises contacting the composition with skin, outer ear,eye, or a combination thereof.

In some embodiments, the composition comprising one or more nucleasepolypeptides disclosed herein is used to contact a food machinerycomprising DNA substrate and to clean the food machinery. In someembodiments, the composition comprising one or more nucleasepolypeptides disclosed herein is used to contact a surface comprisingDNA substrate and to clean the surface. In some embodiments, thecomposition comprising one or more nuclease polypeptides disclosedherein is used to contact a paper machine comprising DNA substrate andto clean the paper machine.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in furtherdetail in the following examples, which are not in any way intended tolimit the scope of the present disclosure.

Protocols used for testing nuclease variants in the following examplesare provided below.

PicoGreen Assay

-   -   Reagents used in the PicoGreen array included:    -   37° C. buffer: 50 mM Tris-HCl, pH8.30, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   54° C. buffer: 50 mM Tris-HCl, pH8.78, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   Enzyme dilution buffer: 37 C buffer with 0.1-1 mg/mL BSA;    -   Substrate stock: Herring Sperm DNA (Promega D1815), 10 mg/mL;    -   37° C. substrate: 4800 ng/mL herring sperm DNA in 37° C. buffer;    -   54° C. substrate: 7200 ng/mL herring sperm DNA in 54° C. buffer;    -   PicoGreen Dye: Thermal Fisher (P7581);    -   PicoGreen reagent: dilute the PicoGreen dye original stock from        vendor 50 times in 50 mM Tris-HCl, pH 7.5 buffer.

Supernatant samples were diluted in enzyme dilution buffer atappropriate dilutions. For the 37° C. assay, 30 uL of 37 C substrate wasmixed with 10 uL of diluted sample/control, and incubated at 37° C. for1 hour. For the 54° C. assay, 20 uL of 54° C. substrate was mixed with20 uL of diluted sample/control, and incubated at 54 C for 1 hour. Afterincubation, 30 uL of PicoGreen reagent was added. Fluorescence was readat Ex/Em=480/520 nm. PicoGreen dye would bind to non-degraded doublestrand DNA and exhibit fluorescence signal.

DNA degradation percentage (also referred to herein as “DNA degradation%,” typically within 20-60%) was calculated as: (fluorescence of wellswithout enzymes-fluorescence of wells with enzymes)/fluorescence ofwells without enzymes×100%. In some examples, activity ratio wascalculated by DNA degradation percentage at 54° C./37° C.

A260 Assay

Reagents used in the A260 assay included:

-   -   37° C. buffer: 50 mM Tris-HCl, pH8.30, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   54° C. buffer: 50 mM Tris-HCl, pH8.78, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   Enzyme dilution buffer: 37° C. buffer with 0.1-1 mg/mL BSA;    -   Substrate stock: Herring Sperm DNA (Promega D1815), 10 mg/mL;    -   37° C. substrate: 0.4 mg/mL herring sperm DNA in 37° C. buffer;    -   54° C. substrate: 0.4 mg/mL herring sperm DNA in 54° C. buffer;    -   4% perchloric acid.

Supernatant samples were diluted in enzyme dilution buffer atappropriate dilutions. In shallow transparent 96-well plate, reactionwas set up in each well. For 37° C. assay, mix 7 uL of dilutedenzyme/control and 140 uL of 37° C. substrate. For 54° C. assay, mix 21uL of diluted enzyme/control and 140 uL of 54° C. substrate. Assayplates were incubated at 37° C. and 54° C. respectively for 1 hour.After incubation, add 147 uL of 4% perchloric acid to each well andcentrifuge at 3200 g at 10° C. for 15 minutes. 230 uL of supernatant wastransferred from each plate to UV transparent plate, and absorbance wasread at 260 nm. Degraded DNA exhibited increased Absorbance 260 signalcompared to non-degraded DNA, and higher A260 values corresponded tohigh nuclease activities. In some examples, activity ratio wascalculated by increased Abs260 at 54° C./37° C.

Fluorescence Resonance Energy Transfer (FRET) Assay—Nuclease DetectionSystem

The FRET assay was modified from manufacture manual, IDT DNaseAlert™Substrate. Reagents used in the assay included:

-   -   Substrate: DNaseAlert™ Substrate (IDT, cat #11-04-02-04). 1 mL        nuclease-free water was added to the substrate bottle, substrate        concentration=2 mM;    -   37° C. buffer: 50 mM Tris-HCl, pH8.30, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   54° C. buffer: 50 mM Tris-HCl, pH8.78, 1 mM MgCl₂, with or        without 0.1-1 mg/mL BSA; or 50 mM Bis-Tris Propane, pH8, 1 mM        MgCl₂, with or without 0.1-1 mg/mL BSA;    -   Enzyme dilution buffer: 37° C. buffer with 0.1-1 mg/mL BSA.

In 96-well half area plates, 5 uL of DNA substrate (2 uM) was mixed with40 uL of BTP buffer and pre-heated at 37° C. or 54° C. for 10 minutes.Enzyme samples were diluted in enzyme dilution buffer around 20-40times. 5 uL of diluted samples was added to substrate solution and readat 37° C. or 54° C. for 15 minutes, excitation=536 nm, emission=566 nm.Slope was calculated by fitting the first three points and obtainedRFU/min.

It was expected that higher slope corresponded to higher nucleaseactivity. If the slope was too high or too low and outside of the linearrange, sample dilution were adjusted and assay were performed again. Insome examples, activity ratio was calculated by slope at 54° C./37° C.

Protein Quantification by LabChip and ELISA

Protein quantifications were performed by LabChip or the enzyme-linkedimmunosorbent assay (ELISA) according to the manufacture's protocol orstandard protocol.

Protocol was slightly modified from the protocol provided by CaliperLifeSciences. LabChip GXII Protein Assay Quick Guide (CaliperLifeSciences, Hopkinton, Mass.) was followed for high sensitivitysamples. Briefly, samples were prepared as below: (1) denaturingsolution was prepared by adding 24.5 uL β-mercaptoethanol to 700 uL ofProtein Express Sample Buffer; (2) 10 uL of protein sample was added to14 uL denaturing solution. Samples could be prepared in 96-well plate;(3) 12 uL of Protein Express Ladder was transferred to microcentrifugetube, but did not add denaturing solution; (4) samples and ladder weredenatured at 100° C. for 5 minutes; (5) 64 uL water was added to samplesand 120 uL water was added to ladder, and centrifuged at 1200 g for 2minutes to get rid of bubbles; (6) 120 uL of ladder was transferred tothe provided Ladder tube; and (7) LabChip measurement was performedaccording to manufacture protocol.

Protein Quantification was conducted by ELISA using standard protocol.

Example 1 Test Nuclease Variants by PicoGreen Assay

PicoGreen assay was used to select desired nuclease variants generatedby GSSM (also referred to herein as “GSSM variants”). DNA degradationpercentage 54° C./37° C. ratio of the nuclease variants relative to thatof the parent nuclease, and expression level of the nuclease variantswere used as selection criteria.

Enzyme expression level was determined by enzyme dilution before theactivity assay at 37° C. Enzyme expression is denoted with “x”, and theexpression level is defined as shown below in Table 1.

TABLE 1 Determination of protein expression level Dilution Factor (DF)Range Expression Level DF >= 20000 xxxx 1000 < DF < 20000 xxx DF 500 =<DF <= 1000 xx DF < 500 x

54° C. Specific Activity Index was calculated by DNA degradation%×dilution factor at 54° C. divided by enzyme concentration. Variousnuclease variants generated by GSSM were tested, and the assay resultsare shown in Table 2. Variants VAR002, VAR003, VAR030, VAR033, VAR035,VAR036, and VAR037 were selected since their activity at 54° C. wasimproved compared to the parent nuclease and they were expressed at orabove a desired level.

TABLE 2 Properties of nuclease variants 54° C. DNA 54° C. Specificdegradation % DNA Specific Activity 54° C./37° C. degradation % ActivitySequence/ compared Expression relative to Name 54° C./37° C. IndexMutation(s) to parent Level parent Parent 0.128 SEQ ID NO: 1 NucleaseVAR001 0.130 24672 E230L 102%  xxxx  90% VAR002 0.187 11348 L114F 47%xxxx 129% VAR003 0.172 13973 L114F 58% xxxx 119% VAR004 0.145 20003E260R 83% xxx 100% VAR005 0.132 32956 E230M 137%  xxx  92% VAR006 0.14222816 D246P 95% xxx  98% VAR007 0.140 34175 S161R 142%  xxx  97% VAR0080.184 N54S, E230M xx 127% VAR009 0.180 15832 T227H 66% xx 125% VAR0100.191 V226K xx 132% VAR011 0.164 20000 E230R 83% xx 114% VAR012 0.171G263A xx 118% VAR013 0.139 V226K xx  96% VAR014 0.142 G263A xx  98%VAR015 0.160 3417 N127S 14% xx 111% VAR016 0.165 P84V xx 114% VAR0170.196 4514 D83E 19% xx 136% VAR018 0.128 D28G, V45T x  89% VAR019 0.146M262V x 101% VAR020 0.144 A190K x 100% VAR021 0.206 P84N x 142% VAR0220.218 I44R x 151% VAR023 0.133 G256S x  92% VAR024 0.205 A73M, P179L x141% VAR025 0.210 I44R x 145% VAR026 0.275 Q135E x 190% VAR027 0.30784913 A60P 15% xx 239% VAR028 0.115 432703 V247I 79% xxx  90% VAR0290.101 184464 G263K 34% xx  79% VAR030 0.145 585026 G119N 107%  xxxx 113%VAR031 0.133 644722 S161E 118%  xxxx 103% VAR032 0.337 186139 P72N 34%xx 263% VAR033 0.148 819361 D107N 149%  xxxx 115% VAR034 0.309 98026P84L 18% xx 241% VAR035 0.172 884237 S74N, Q141R 161%  xxxx 134% VAR0360.141 1165338 T82R 213%  xxxx 110% VAR037 0.161 1096140 G75R 200%  xxxx126% VAR038 0.146 1024096 G75R 187%  xxxx 114%

Example 2 Test Nuclease Variants Generated by TMSCA

FRET assay was used to select desired nuclease variants generated byTMSCA (also referred to herein as “TMSCA mutants”). 54° C./37° C.reaction rate ratio (>1) and enzyme dilution factor (>20) were used asselection criteria. Selected variants had improved activity at 54° C.compared to the parent nuclease. The results of the FRET assay are shownin Table 3.

TABLE 3 Properties of nuclease variants generated by TMSCA 54° C./37° C.Reaction Speed Name Sequence/Mutations Ratio Parent nuclease SEQ ID NO:1 0.62 VAR045 T82R, L114F 1.00 VAR046 T82R, L114F, G119N 1.11 VAR047T82R 0.56 VAR048 T82R, G119N 0.64 VAR049 L114F 0.76 VAR050 L114F, G119N0.75 VAR051 G119N 0.70 VAR052 G75R, T82R, L114F 1.25 VAR053 G75R, T82R,L114F, G119N 1.62 VAR054 G75R, T82R 0.75 VAR055 G75R, L114F 0.83 VAR056G75R, L114F, G119N 1.14 VAR057 G75R 0.68 VAR058 G75R, G119N 0.65 VAR060S74N, G75R, T82R, G119N, Q141R 3.37 VAR061 S74N, T82R, L114F, Q141R 2.06VAR062 S74N, T82R, L114F, G119N, Q141R 1.98 VAR063 S74N, T82R, Q141R1.24 VAR064 S74N, T82R, G119N, Q141R 1.39 VAR065 S74N, L114F, Q141R 1.21VAR066 S74N, L114F, G119N, Q141R 1.27 VAR067 S74N, Q141R 0.80 VAR068S74N, G75R, T82R, L114F, Q141R 3.55 VAR069 S74N, G75R, T82R, L114F,G119N, 2.70 Q141R VAR070 S74N, G75R, T82R, Q141R 2.90 VAR071 S74N, G75R,L114F, Q141R 3.27 VAR072 S74N, G75R, Q141R 2.87 VAR073 T82R, D107N 1.57VAR074 G75R, T82R, D107N, 1.48 VAR075 D107N 0.39 VAR076 G75R, T82R,G119N 0.97 VAR077 T82R, D107N, G119N 1.44 VAR078 G75R, T82R, D107N,G119N 1.16 VAR079 D107N, G119N 1.72 VAR080 G75R, D107N, G119N 1.28VAR081 S74N, T82R, D107N, Q141R 1.64 VAR082 S74N, G75R, T82R, D107N,Q141R 1.96 VAR083 S74N, G75R, D107N, Q141R 1.78 VAR084 S74N, G119N,Q141R 1.19 VAR085 S74N, G75R, G119N, Q141R 2.77 VAR086 S74N, T82R,D107N, G119N, Q141R 1.64 VAR087 S74N, G75R, T82R, D107N, G119N, 2.19Q141R VAR088 S74N, D107N, G119N, Q141R 1.69 VAR089 S74N, G75R, D107N,G119N, Q141R 1.49 VAR090 T82R, D107N, L114F 1.87 VAR091 G75R, T82R,D107N, L114F 1.75 VAR092 D107N, L114F 1.60 VAR093 G75R, D107N, L114F1.47 VAR094 G75R, D107N 1.59 VAR095 T82R, D107N, L114F, G119N 1.57VAR096 D107N, L114F, G119N 1.59 VAR097 G75R, D107N, L114F, G119N 1.57VAR098 S74N, T82R, D107N, L114F, Q141R 1.95 VAR099 S74N, G75R, T82R,D107N, L114F, 1.95 Q141R VAR100 S74N, D107N, L114F, Q141R 2.03 VAR101S74N, G75R, D107N, L114F, Q141R 1.43 VAR102 S74N, D107N, Q141R 1.45VAR103 S74N, G75R, L114F, G119N, Q141R 2.76 VAR104 S74N, T82R, D107N,L114F, G119N, 2.04 Q141R VAR105 S74N, G75R, T82R, D107N, L114F, 1.82G119N, Q141R VAR106 S74N, D107N, L114F, G119N, Q141R 4.95

Example 3 Test Nuclease Variants by A260 Assay

A number of nuclease variants generated by TMSCA were tested using A260assay. The results of the assay are shown in Table 4. The selectioncriteria used were: activity 54° C./37° C. at pH7 and pH8 both >1; at37° C., pH7/pH8>0.9; and at 54° C., pH7/pH8>0.5. Selected hits haveimproved activity than the parent at pH7 and pH8, at 54° C.; at 37° C.and 54° C., activity at pH7.

TABLE 4 A260 assay results of TMSCA nuclease variants pH 8 pH 7 37° C.54° C. 54° C./37° C. 54° C./37° C. pH 7/pH 8 pH 7/pH 8 VAR046 0.78 0.230.81 0.24 VAR053 1.04 0.62 0.95 0.57 VAR060 2.56 1.87 1.08 0.79 VAR0612.70 1.29 1.07 0.51 VAR062 2.40 1.28 1.07 0.57 VAR064 1.25 0.86 0.900.62 VAR065 1.10 0.44 1.08 0.43 VAR066 2.34 0.70 1.08 0.33 VAR068 2.912.12 1.12 0.82 VAR069 3.48 2.75 1.12 0.88 VAR070 2.80 2.05 1.17 0.86VAR071 3.02 2.05 1.18 0.80 VAR072 2.51 1.51 1.06 0.63 VAR077 0.56 0.420.16 0.12 VAR078 2.14 1.30 0.69 0.42 VAR085 1.63 1.04 1.09 0.70 VAR0900.48 0.58 0.22 0.27 VAR091 1.36 0.97 0.30 0.21 VAR093 0.73 0.94 0.360.46 VAR095 0.54 0.74 0.21 0.28 VAR103 3.46 2.78 1.01 0.81 Seq 1 0.230.14 0.43 0.27

Example 4 Temperature Profiling of Nuclease Variants Generated by TMSCA

A number of nuclease variants generated by TMSCA were tested fortemperature profiling. The test results are shown in Table 5(PC=positive control, and NC=negative control) and FIG. 2. In Table 5,the temperature (“temp”) under which the nuclease variants were testedis provided at the most left column.

TABLE 5 A260 assay results normalized by maximum activity % for TMSCAnuclease variants Temp VAR060 VAR061 VAR062 VAR068 VAR069 VAR070 VAR071VAR072 VAR085 VAR103 POS NC 70 15 13 11 29 28.3 15.9 17.6 11.0 11.8 16.010.3 68.7 18 15 13 32 33.3 18.8 22.0 130 141 198 10.0 65.9 32 25 24 4142.4 29.9 33.2 224 239 297 10.1 61.8 52 43 50 56 60.7 49.9 50.8 460 452451 10.8 57 83 59 72 84 92.9 71.5 67.8 688 677 740 14.0 53 96 85 95 9899.6 94.3 96.7 926 974 973 21.4 50.3 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0 100.0 31.0 49 91 100 94 90 86.2 93.1 92.6 89.4 96.590.2 43.6 47.7 83 98 86 83 76.6 85.5 85.0 80.2 85.5 78.8 55.8 45.3 74 9574 73 63.4 79.2 74.2 73.3 77.4 66.8 71.8 41.5 53 76 52 48 40.1 59.6 48.650.5 55.9 43.7 91.1 37.2 37 54 36 31 25.8 43.7 30.9 36.3 39.4 26.9 98.533.6 29.0 40.0 27.0 21.0 18.3 33.5 20.5 27.4 30.0 17.8 99.2 31.2 23.033.0 22.0 16.0 14.3 26.3 15.1 22.2 24.7 13.7 100.0 30 21.0 29.0 20.014.0 12.1 23.9 12.6 19.2 21.4 11.6 97.7

Example 5 pH Profiling of Nuclease Variants at 37° C.

A number of nuclease variants generated by TMSCA were tested for pHprofiling at 37° C. The test results are shown in Table 6 and FIG. 3.

TABLE 6 A260 reading results normalized to maximum activity for nucleasevariants pH VAR060 VAR061 VAR062 VAR068 VAR069 VAR070 VAR071 VAR072VAR085 VAR103 PC NC 6.3 76.4 79.6 77.9 85.1 83.0 82.1 81.0 82.4 82.286.2 40.8 6.7 95.3 97.9 95.4 97.8 98.5 99.2 94.8 94.3 95.4 97.5 63.1 7.0100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100 75.9 7.5 91.291.0 92.3 89.6 88.9 88.2 85.9 87.1 87.8 87.6 85.9 8.0 78.9 82.2 85.579.7 80.1 76.3 76.3 77.1 79.1 76.0 96.7 8.5 71.4 74.4 80.6 77.8 73.666.9 67.5 70.1 71.0 66.4 98.0 9.0 63.7 72.9 82.3 66.5 69.5 59.8 68.365.7 68.1 68.5 85.2 9.3 58.5 74.6 82.3 65.9 68.2 55.4 68.0 63.2 65.867.9 100.0 10.0 56.6 58.3 64.5 59.9 61.1 56.6 53.4 60.3 62.3 54.3 67.9

Example 6 Nuclease Pretreatment of an Alpha Amylase Broth

Five lead variants VAR060, VAR062, VAR070, VAR072, and VAR085 wereselected. For each of these five lead variants, heat kill of an alphaamylase broth was performed (see FIG. 4). Nuclease was added during thetwo hour ramping time of heat kill process.

The pretreatment results for the lead variants were normalized bynuclease concentration. Each variant was dosed at 3 levels in duplicate:0.23 ppb, 0.45 ppb, and 0.9 ppb, which are equivalent to 250, 500, and1000 U/L for Benzonase® endonuclease, respectively. Controls used foreach dosing included: positive (the nuclease having the sequence of SEQID NO: 1), negative (vector control, diluted identically to parent), andBenzonase®. The un-normalized and normalized results of pretreatment areshown in FIG. 5, respectively. Among all the lead variants tested,VAR070 showed overall greatest viscosity reduction at each dose.

As shown in the examples disclosed herein, a number of nuclease variantshaving single point mutations have higher activity ratio 54 C°/37 C°compared to the parent nuclease while keeping similar specific activity.Also a number of nuclease variants having combinations of pointmutations have an optimal temperature between 45 C°-55 C° (compared to37 C° for the parent nuclease), and an optimal pH of about 6.5 comparedto pH 8 for the parent nuclease.

The foregoing description and examples detail certain preferredembodiments of the description and describes the best mode contemplatedby the inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the description may bepracticed in many ways and the description should be construed inaccordance with the appended claims and any equivalents thereof.Although the present application has been described in detail above, itwill be understood by one of ordinary skill in the art that variousmodifications can be made without departing from the spirit of thedescription.

In the present application, the use of the singular can include theplural unless specifically stated otherwise or unless, as will beunderstood by one of skill in the art in light of the presentdisclosure, the singular is the only functional embodiment. Thus, forexample, “a” can mean more than one, and “one embodiment” can mean thatthe description applies to multiple embodiments. Additionally, in thisapplication, “and/or” denotes that both the inclusive meaning of “and”and, alternatively, the exclusive meaning of “or” applies to the list.Thus, the listing should be read to include all possible combinations ofthe items of the list and to also include each item, exclusively, fromthe other items. The addition of this term is not meant to denote anyparticular meaning to the use of the terms “and” or “or” alone. Themeaning of such terms will be evident to one of skill in the art uponreading the particular disclosure.

All references cited herein including, but not limited to, published andunpublished patent applications, patents, text books, literaturereferences, and the like, to the extent that they are not already, arehereby incorporated by reference in their entirety. To the extent thatone or more of the incorporated literature and similar materials differfrom or contradict the disclosure contained in the specification,including but not limited to defined terms, term usage, describedtechniques, or the like, the specification is intended to supersedeand/or take precedence over any such contradictory material.

The term “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps.

What is claimed is: 1-60. (canceled)
 61. A synthetic or recombinantpolypeptide comprising an amino acid sequence having at least 80%sequence identity to SEQ ID NO: 1, wherein the polypeptide has nucleaseactivity, and wherein the polypeptide comprises one or more mutations ofE230L, L114F, E260R, E230M, D246P, S161R, N54S, T227H, E230R, G263A,G119N, V226K, N127S, P84V, D83E, D28G, V45T, M262V, A190K, P84N, I44R,G256S, A73M, P179L, Q135E, A60P, V247I, G263K, S161E, P72N, P84L, S74N,T82R, G75R, Q141R, and D107N.
 62. The polypeptide of claim 61, whereinthe polypeptide comprises a mutation or a combination of mutationsselected from the group consisting of (T82R, L114F); (T82R, L114F,G119N); (T82R) (T82R, G119N); (L114F); (L114F, G119N); (G119N); (G75R,T82R, L114F, G119N); (G75R, T82R, L114F, G119N); (G75R, T82R); (G75R,L114F); (G75R, L114F, G119N); (S74N, G75R, T82R, G119N, Q141R); (S74N,T82R, L114F, G119N); (S74N, T82R, L114F, G119N, Q141R); (S74N, T82R,Q141R); (S74N, T82R, G119N, Q141R); (S74N, L114F, Q141R); (S74N, L114F,G119N, Q141R); (S74N, Q141R); (S74N, G75R, T82R, L114F, Q141R); (S74N,G75R, T82R, L114F, G119N, Q141R); (S74N, G75R, T82R, Q141R); (S74N,G75R, L114F, Q141R); (S74N, G75R, Q141R); (T82R, D107N); (G75R, T82R,D107N); (D107N); (G75R, T82R, G119N); (T82R, D107N, G119N); (G75R, T82R,D107N, G119N); (D107N, G119N); (G75R, D107N, G119N); (S74N, T82R, D107N,Q141R); (S74N, G75R, T82R, D107N, Q141R); (S74N, G75R, D107N, Q141R);(S74N, G119N, Q141R); (S74N, G75R, G119N, Q141R); (S74N, T82R, D107N,G119N, Q141R); (S74N, G75R, T82R, D107N, G119N, Q141R); (S74N, D107N,G119N, Q141R); (S74N, G75R, D107N, G119N, Q141R); (T82R, D107N, L114F);(G75R, T82R, D107N, L114F); (D107N, L114F); (G75R, D107N, L114F); (G75R,D107N); (T82R, D107N, L114F, G119N); (D107N, L114F, G119N); (G75R,D107N, L114F, G119N); (S74N, T82R, D107N, L114F, Q141R); (S74N, G75R,T82R, D107N, L114F, Q141R); (S74N, D107N, L114F, Q141R); (S74N, G75R,D107N, L114F, Q141R); (S74N, D107N, Q141R); (S74N, G75R, L114F, G119N,Q141R); (S74N, T82R, D107N, L114F, G119N, Q141R); (S74N, G75R, T82R,D107N, L114F, G119N, Q141R); (S74N, D107N, L114F, G119N, Q141R); (S74N,G75R, D107N, L114F, G119N, Q141R); and (G75R, T82R, D107N, L114F,G119N).
 63. The polypeptide of claim 61, wherein the amino acid sequenceof the polypeptide differs from the amino acid sequence of SEQ ID NO: 1for comprising one or more mutations of E230L, L114F, E260R, E230M,D246P, S161R, N54S, T227H, E230R, G119N, V226K, G263A, N127S, P84V,D83E, D28G, V45T, M262V, A190K, P84N, I44R, G256S, A73M, P179L, Q135E,A60P, V247I, G263K, S161E, P72N, P84L, S74N, T82R, G75R, Q141R, andD107N.
 64. The polypeptide of claim 61, wherein the polypeptide is morethermotolerant compared to the nuclease having the sequence of SEQ IDNO:
 1. 65. The polypeptide of claim 61, wherein the nuclease activity ofthe polypeptide is at least 5% higher than that of the nuclease havingthe sequence of SEQ ID NO: 1 at 10° C. to 70° C.
 66. The polypeptide ofclaim 61, the optimal temperature of the polypeptide is between 40° C.to 60° C.
 67. The polypeptide of claim 61, wherein optimal pH of thepolypeptide is between pH 4 to pH
 11. 68. The polypeptide of claim 61,wherein the polypeptide comprises no signal sequence.
 69. Thepolypeptide of claim 61, wherein the polypeptide comprises a signalsequence, and wherein the signal sequence is a heterologous sequence ora native signal sequence.
 70. A composition comprising the polypeptideof claim
 61. 71. The composition of claim 61, wherein the composition isa reaction mixture, a detergent composition, a detergent additive, afood, a food supplement, a feed supplement, a feed, a pharmaceuticalcomposition, a fermentation product, a fermentation intermediate, afermentation downstream reaction mixture, or a combination thereof. 72.The composition of claim 61, wherein the reaction mixture is for proteinexpression or purification.
 73. A synthetic or recombinant nucleic acidthat encodes the polypeptide of claim
 61. 74. An expression vectorcomprising the nucleic acid of claim
 73. 75. The expression vector ofclaim 74, wherein the expression vector comprises a viral vector, aplasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage, anartificial chromosome, or a combination thereof.
 76. A recombinant cellcomprising the polypeptide of claim 61, the nucleic acid of claim 73,the expression vector of claim 74, or a combination thereof.
 77. Therecombinant cell of claim 76, wherein the nucleic acid is a part of achromosome of the recombinant cell, and wherein the cell is a bacterialcell, a mammalian cell, a fungal cell, a yeast cell, an insect cell, ora plant cell.
 78. A method of producing a recombinant polypeptide havingnuclease activity, comprising: expressing the nucleic acid of claim 61under conditions that allow expression of the polypeptide, therebyproducing recombinant polypeptide having nuclease activity, wherein thenucleic acid is operably linked to a promoter.
 79. The method of claim78, wherein the nucleic acid is present in an expression vector.
 80. Themethod of claim 78, wherein the nucleic acid is present an in vitroexpression system.
 81. The method of claim 78, wherein the nucleic acidis present in a host cell to allow expression of the polypeptide, andwherein the host cell is a cell from an organism selected from the groupconsisting of Pichia pastoris (Komagataella pastoris), Bacillussubtilis, Pseudomonas fluorescens, Myceliopthora thermophile fungus,Tricodermea reesei, Escherichia coli, Bacillus licheniformis,Aspergillus niger, Schizosaccharomyces pombe, and Sacaramycescerevisiae.
 82. The method of claim 78, wherein the nucleic acid is aRNA molecule.
 83. A method for degrading a polynucleotide, comprisingcontacting a polynucleotide molecule with the polypeptide of claim 61,thereby degrading the polynucleotide molecule.
 84. The method of claim83, wherein the polynucleotide molecule is a DNA molecule or a RNAmolecule; and wherein the contacting occurs at pH 4 to pH 1 land atabout 10° C. to about 70° C.
 85. A method for washing an object,comprising contacting a composition comprising the polypeptide of claim61 with the object under the conditions sufficient for said washing. 86.A method for degrading DNA or RNA during protein production, comprisingculturing a host cell, wherein the host cell comprises a nucleic acidencoding a protein of interest; and expressing the polypeptide of claim61 under conditions that allow degradation of DNA or RNA by thepolypeptide.
 87. The method of claim 86, wherein the host cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, a plantcell, or an insect cell.
 88. The method of claim 86, wherein thepolypeptide is expressed from an expression vector present in the hostcell or the polypeptide is encoded by a nucleic acid sequence in achromosome of the host cell.
 89. The method of claim 86, wherein thepolypeptide is expressed by cells that do not express the protein ofinterest.
 90. The method of claim 86, wherein expression of one or moreof the protein of interest and/or the polypeptide is inducible ornon-inducible.
 91. A reaction mixture, comprising: the polypeptide ofclaim 61; one or more nucleic acid molecules; and an aqueous solutionwherein the polypeptide hydrolyzes the one or more nucleic acidmolecules.
 92. The reaction mixture of claim 91, wherein the one or morenucleic acid molecules comprise single-stranded DNA molecules,double-stranded DNA molecules, single-stranded RNA molecules,double-stranded RNA molecules, or any combination thereof.
 93. Thereaction mixture of claim 91, wherein the one or more nucleic acidmolecules are from a host cell for protein production.
 94. The reactionmixture of claim 91, wherein the polypeptide is expressed in a host cellselected from the group consisting of bacterial cell, a mammalian cell,a fungal cell, a yeast cell, a plant cell, and an insect cell.
 95. Thereaction mixture of claim 91, wherein the nucleic acid and/orpolypeptide is expressed by in vitro transcription or translation. 96.The reaction mixture of claim 91, wherein the reaction mixture has atemperature at about 10° C. to about 70° C.
 97. The reaction mixture ofclaim 91, wherein the reaction mixture is at about pH 4 to about pH 11.98. The reaction mixture of claim 91, wherein the aqueous solution is adetergent composition, a detergent additive, a food, a food supplement,a feed supplement, a feed, a pharmaceutical composition, a fermentationproduct, a fermentation intermediate, a fermentation downstream reactionmixture, a product from protein production process, an intermediate fromprotein production process, or a protein purification solution.
 99. Amethod for degrading DNA or RNA in a protein production mixture,comprising culturing a host cell, wherein the host cell comprises anucleic acid encoding a protein of interest; and expressing thepolypeptide of claim 61 under conditions that allow degradation of DNAor RNA by the polypeptide.
 100. The method of claim 99, wherein the hostcell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell,a plant cell, or an insect cell.
 101. The method of claim 99, whereinthe polypeptide is expressed from an expression vector present in thehost cell or the polypeptide is encoded by a nucleic acid sequence in achromosome of the host cell.
 102. The method of claim 99, wherein thepolypeptide is expressed by cells that do not express the protein ofinterest.
 103. The method of claim 99, wherein expression of one or moreof the protein of interest and/or the polypeptide is inducible ornon-inducible.
 104. A method for degrading DNA or RNA during proteinproduction, comprising: culturing a host cell, wherein the host cellcomprises a nucleic acid encoding a protein of interest; and adding apolypeptide of claim 61 under conditions that allow degradation of DNAor RNA by the polypeptide.
 105. The method of claim 104, wherein thehost cell is a bacterial cell, a mammalian cell, a fungal cell, a yeastcell, a plant cell, or an insect cell.